Confocal Microscopy List

Nikon C1si?

Classic

List

Threaded

40 messages Options

Craig Brideau

Re: Nikon C1si?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It doesn't seem to deal with depth though? It only detects in X-Y, so
can it generate optical slices?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The PARISS system uses a Retiga 2000R cooled CCD camera. It is far more
> sensitive and accurate than any confocal microscope.
> You can detect things in that system that can not be observed with a
> confocal spectral system. It is a great system in my opinion.
> Bob
>
> Robert M. Zucker, PhD
> U.S. Environmental Protection Agency
> Office of Research and Development
> National Health and Environmental Effects Research Laboratory.
> Telephone: 919-541-1585 Fax: 919-541-4017
> e-mail: [hidden email]
>
> Mail address: Reproductive Toxicology Division, MD 67
> 2525 E.NC Highway 54
> Research Triangle Park, North Carolina, 27711
>
> Shipping address: 2525 E.NC Highway 54
> Durham, NC, 27713
>
>
>
>
> Craig Brideau
> <craig.brideau@G
> MAIL.COM> To
> Sent by: [hidden email]
> Confocal cc
> Microscopy List
> <CONFOCAL@LISTSE Subject
> RV.BUFFALO.EDU> Re: Nikon C1si?
>
>
> 10/05/2007 06:19
> PM
>
>
> Please respond
> to
> Confocal
> Microscopy List
> <CONFOCAL@LISTSE
> RV.BUFFALO.EDU>
>
>
>
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Will this system have the same sort of sensitivity as a multi-anode
> PMT system like the C1si? This PARISS system seems to use a CCD
> camera as its detector, which will not be as sensitive, if I
> understand correctly?
>
> Craig
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Criag
> > If spectral analysis is the endpoint you may want to consider the
> PARISS
> > system from Lightform. ( http://www.lightforminc.com/) that can be
> > placed on a widefield microscope.
> > We are using the PARISS system to obtain spectral data --it has 1nm
> > resolution and has a spectrum from 400-900nm. I find it to be a great
> > asset in the laboratory,
> > It is far more sensitive and accurate that any confocal system that I
> > have seen.
> >
> > Contact Jeremy Lerner for details on his system.
> > best wishes
> > Bob
> >
> >
> >
> >
> >
> >
> >
> >
> > LightForm, Inc.,
> > 601 Route 206, Suite 26-479
> > Hillsborough NJ 08844
> > Tel: (908)281 9098
> > Email: [hidden email]
> >
> >
> >
> >
> >
> >
> > .
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585 Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> > Craig Brideau
> > <craig.brideau@G
> > MAIL.COM>
> To
> > Sent by: [hidden email]
> > Confocal
> cc
> > Microscopy List
> > <CONFOCAL@LISTSE
> Subject
> > RV.BUFFALO.EDU> Re: Nikon C1si?
> >
> >
> > 10/05/2007 05:48
> > PM
> >
> >
> > Please respond
> > to
> > Confocal
> > Microscopy List
> > <CONFOCAL@LISTSE
> > RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Thanks for your input! As in everything, there's a trade-off. Still,
> > for us the spectral data will be worth it.
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Craig
> > > The efficiency of detection is much less in the spectral mode due to
> > the
> > > design of the multianode detector. In addition as Kurt Thorn said
> the
> > > light is split into a number of different bandpass channels instead
> of
> > > being detected by only one channel which also limits the detection
> in
> > > each channel.
> > > Nikon compensates for this decreased light by increasing the pixel
> > dwell
> > > time. However photons are photons. With less photons you will get a
> > > noisier image.
> > > The spectral detection of any confocal system using a multianode
> > > detector will not be as good as a PMT designed for good sensitivity
> > and
> > > low light detection. It will produce nosier images but if there is
> > > enough light you will be able to determine a valuable spectrum which
> > can
> > > be used for scientific experiments and to manipulate images. Like
> > > everything with confocal microscopy there is trade-offs and no
> perfect
> > > system.
> > > Best wishes.
> > > Bob
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by: [hidden email]
> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/04/2007 02:49
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > I was wondering if you saw this decreased throughput in spectral
> mode
> > > only, or also in conventional detection mode? The spectral mode
> works
> > > with a multi-anode PMT, which has a lower quantum efficiency than a
> > > conventional PMT. From what I have seen of the design, it also has
> > > the option to use a conventional PMT system with the device, and I
> am
> > > wondering if you have also tried that mode of operation?
> > >
> > > Thanks,
> > >
> > > Craig
> > >
> > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > One of the major limitations that we have seen with this unit and
> > also
> > > > with the Zeiss meta 510 is the decreased light throughput . This
> > > > creates images that are noisier than conventional confocal
> > > microscopes.
> > > > In our hands it appears you will need to have a bright sample to
> > make
> > > > the spectral system work properly. ,
> > >
> >
>

Zucker.Robert

Re: Nikon C1si?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
you are correct.
Currently the PARISS only detects X-Y--not depth.
however, most data that I have seen from confocal spectral systems are
only a single XY slice. they usually are not used to do a 3D
reconstruction in spectral mode.
bob
.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

Craig Brideau
<craig.brideau@G
MAIL.COM> To
Sent by: [hidden email]
Confocal cc
Microscopy List
<CONFOCAL@LISTSE Subject
RV.BUFFALO.EDU> Re: Nikon C1si?

10/05/2007 06:44
PM

Please respond
to
Confocal
Microscopy List
<CONFOCAL@LISTSE
RV.BUFFALO.EDU>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It doesn't seem to deal with depth though? It only detects in X-Y, so
can it generate optical slices?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
more

> sensitive and accurate than any confocal microscope.
> You can detect things in that system that can not be observed with a
> confocal spectral system. It is a great system in my opinion.
> Bob
>
> Robert M. Zucker, PhD
> U.S. Environmental Protection Agency
> Office of Research and Development
> National Health and Environmental Effects Research Laboratory.
> Telephone: 919-541-1585 Fax: 919-541-4017
> e-mail: [hidden email]
>
> Mail address: Reproductive Toxicology Division, MD 67
> 2525 E.NC Highway 54
> Research Triangle Park, North Carolina, 27711
>
> Shipping address: 2525 E.NC Highway 54
> Durham, NC, 27713
>
>
>
>
> Craig Brideau
> <craig.brideau@G
> MAIL.COM>

To
> Sent by: [hidden email]
> Confocal
cc
> Microscopy List
> <CONFOCAL@LISTSE
Subject

> RV.BUFFALO.EDU> Re: Nikon C1si?
>
>
> 10/05/2007 06:19
> PM
>
>
> Please respond
> to
> Confocal
> Microscopy List
> <CONFOCAL@LISTSE
> RV.BUFFALO.EDU>
>
>
>
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Will this system have the same sort of sensitivity as a multi-anode
> PMT system like the C1si? This PARISS system seems to use a CCD
> camera as its detector, which will not be as sensitive, if I
> understand correctly?
>
> Craig
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Criag
> > If spectral analysis is the endpoint you may want to consider the
> PARISS
> > system from Lightform. ( http://www.lightforminc.com/) that can be
> > placed on a widefield microscope.
> > We are using the PARISS system to obtain spectral data --it has 1nm
> > resolution and has a spectrum from 400-900nm. I find it to be a

great
> > asset in the laboratory,
> > It is far more sensitive and accurate that any confocal system that
I

> > have seen.
> >
> > Contact Jeremy Lerner for details on his system.
> > best wishes
> > Bob
> >
> >
> >
> >
> >
> >
> >
> >
> > LightForm, Inc.,
> > 601 Route 206, Suite 26-479
> > Hillsborough NJ 08844
> > Tel: (908)281 9098
> > Email: [hidden email]
> >
> >
> >
> >
> >
> >
> > .
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585 Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> > Craig Brideau
> > <craig.brideau@G
> > MAIL.COM>
> To
> > Sent by: [hidden email]
> > Confocal
> cc
> > Microscopy List
> > <CONFOCAL@LISTSE
> Subject
> > RV.BUFFALO.EDU> Re: Nikon C1si?
> >
> >
> > 10/05/2007 05:48
> > PM
> >
> >
> > Please respond
> > to
> > Confocal
> > Microscopy List
> > <CONFOCAL@LISTSE
> > RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Thanks for your input! As in everything, there's a trade-off.

Still,

> > for us the spectral data will be worth it.
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Craig
> > > The efficiency of detection is much less in the spectral mode due

to
> > the
> > > design of the multianode detector. In addition as Kurt Thorn said
> the
> > > light is split into a number of different bandpass channels
instead
> of
> > > being detected by only one channel which also limits the detection
> in
> > > each channel.
> > > Nikon compensates for this decreased light by increasing the pixel
> > dwell
> > > time. However photons are photons. With less photons you will get
a
> > > noisier image.
> > > The spectral detection of any confocal system using a multianode
> > > detector will not be as good as a PMT designed for good
sensitivity
> > and
> > > low light detection. It will produce nosier images but if there is
> > > enough light you will be able to determine a valuable spectrum
which

> > can
> > > be used for scientific experiments and to manipulate images. Like
> > > everything with confocal microscopy there is trade-offs and no
> perfect
> > > system.
> > > Best wishes.
> > > Bob
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by:

[hidden email]

> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/04/2007 02:49
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > I was wondering if you saw this decreased throughput in spectral
> mode
> > > only, or also in conventional detection mode? The spectral mode
> works
> > > with a multi-anode PMT, which has a lower quantum efficiency than

a
> > > conventional PMT. From what I have seen of the design, it also
has

> > > the option to use a conventional PMT system with the device, and I
> am
> > > wondering if you have also tried that mode of operation?
> > >
> > > Thanks,
> > >
> > > Craig
> > >
> > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > One of the major limitations that we have seen with this unit

and
> > also
> > > > with the Zeiss meta 510 is the decreased light throughput .
This
> > > > creates images that are noisier than conventional confocal
> > > microscopes.
> > > > In our hands it appears you will need to have a bright sample to
> > make
> > > > the spectral system work properly. ,
> > >
> >
>

Craig Brideau

Re: Nikon C1si?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks! We are interested in co-localization, for which we sometimes
need the third dimension. Mainly we want to do spectral unmixing for
multiple dyes and colocalization, so we really do need optical
slicing!

Thanks,

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Craig
> you are correct.
> Currently the PARISS only detects X-Y--not depth.
> however, most data that I have seen from confocal spectral systems are
> only a single XY slice. they usually are not used to do a 3D
> reconstruction in spectral mode.
> bob
> .
>
>
> Robert M. Zucker, PhD
> U.S. Environmental Protection Agency
> Office of Research and Development
> National Health and Environmental Effects Research Laboratory.
> Telephone: 919-541-1585 Fax: 919-541-4017
> e-mail: [hidden email]
>
> Mail address: Reproductive Toxicology Division, MD 67
> 2525 E.NC Highway 54
> Research Triangle Park, North Carolina, 27711
>
> Shipping address: 2525 E.NC Highway 54
> Durham, NC, 27713
>
>
>
>
> Craig Brideau
> <craig.brideau@G
> MAIL.COM> To
> Sent by: [hidden email]
> Confocal cc
> Microscopy List
> <CONFOCAL@LISTSE Subject
> RV.BUFFALO.EDU> Re: Nikon C1si?
>
>
> 10/05/2007 06:44
> PM
>
>
> Please respond
> to
> Confocal
> Microscopy List
> <CONFOCAL@LISTSE
> RV.BUFFALO.EDU>
>
>
>
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> It doesn't seem to deal with depth though? It only detects in X-Y, so
> can it generate optical slices?
>
> Craig
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> more
> > sensitive and accurate than any confocal microscope.
> > You can detect things in that system that can not be observed with a
> > confocal spectral system. It is a great system in my opinion.
> > Bob
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585 Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> > Craig Brideau
> > <craig.brideau@G
> > MAIL.COM>
> To
> > Sent by: [hidden email]
> > Confocal
> cc
> > Microscopy List
> > <CONFOCAL@LISTSE
> Subject
> > RV.BUFFALO.EDU> Re: Nikon C1si?
> >
> >
> > 10/05/2007 06:19
> > PM
> >
> >
> > Please respond
> > to
> > Confocal
> > Microscopy List
> > <CONFOCAL@LISTSE
> > RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Will this system have the same sort of sensitivity as a multi-anode
> > PMT system like the C1si? This PARISS system seems to use a CCD
> > camera as its detector, which will not be as sensitive, if I
> > understand correctly?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Criag
> > > If spectral analysis is the endpoint you may want to consider the
> > PARISS
> > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > placed on a widefield microscope.
> > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > resolution and has a spectrum from 400-900nm. I find it to be a
> great
> > > asset in the laboratory,
> > > It is far more sensitive and accurate that any confocal system that
> I
> > > have seen.
> > >
> > > Contact Jeremy Lerner for details on his system.
> > > best wishes
> > > Bob
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > > LightForm, Inc.,
> > > 601 Route 206, Suite 26-479
> > > Hillsborough NJ 08844
> > > Tel: (908)281 9098
> > > Email: [hidden email]
> > >
> > >
> > >
> > >
> > >
> > >
> > > .
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by: [hidden email]
> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/05/2007 05:48
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Thanks for your input! As in everything, there's a trade-off.
> Still,
> > > for us the spectral data will be worth it.
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > Search the CONFOCAL archive at
> > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Craig
> > > > The efficiency of detection is much less in the spectral mode due
> to
> > > the
> > > > design of the multianode detector. In addition as Kurt Thorn said
> > the
> > > > light is split into a number of different bandpass channels
> instead
> > of
> > > > being detected by only one channel which also limits the detection
> > in
> > > > each channel.
> > > > Nikon compensates for this decreased light by increasing the pixel
> > > dwell
> > > > time. However photons are photons. With less photons you will get
> a
> > > > noisier image.
> > > > The spectral detection of any confocal system using a multianode
> > > > detector will not be as good as a PMT designed for good
> sensitivity
> > > and
> > > > low light detection. It will produce nosier images but if there is
> > > > enough light you will be able to determine a valuable spectrum
> which
> > > can
> > > > be used for scientific experiments and to manipulate images. Like
> > > > everything with confocal microscopy there is trade-offs and no
> > perfect
> > > > system.
> > > > Best wishes.
> > > > Bob
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > e-mail: [hidden email]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > > Craig Brideau
> > > > <craig.brideau@G
> > > > MAIL.COM>
> > > To
> > > > Sent by:
> [hidden email]
> > > > Confocal
> > > cc
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > Subject
> > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > >
> > > >
> > > > 10/04/2007 02:49
> > > > PM
> > > >
> > > >
> > > > Please respond
> > > > to
> > > > Confocal
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > > RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > I was wondering if you saw this decreased throughput in spectral
> > mode
> > > > only, or also in conventional detection mode? The spectral mode
> > works
> > > > with a multi-anode PMT, which has a lower quantum efficiency than
> a
> > > > conventional PMT. From what I have seen of the design, it also
> has
> > > > the option to use a conventional PMT system with the device, and I
> > am
> > > > wondering if you have also tried that mode of operation?
> > > >
> > > > Thanks,
> > > >
> > > > Craig
> > > >
> > > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > > One of the major limitations that we have seen with this unit
> and
> > > also
> > > > > with the Zeiss meta 510 is the decreased light throughput .
> This
> > > > > creates images that are noisier than conventional confocal
> > > > microscopes.
> > > > > In our hands it appears you will need to have a bright sample to
> > > make
> > > > > the spectral system work properly. ,
> > > >
> > >
> >
>

Shalin Mehta

Re: Nikon C1si?

In reply to this post by Craig Brideau

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Craig,

CCDs can convert more photons into electrons in comparison to PMTs. But two major drawbacks are slow readout (due to semi-serial output) and no inherent multiplicative gain (whereby one electron produced by photon is converted into a bunch of photons..) A sufficiently powered PMT will give us a million or more electrons for each electron produced by photoelectric effect. It is easier for downstream electronics to handle large currents. EMCCDs can overcome the problem of multiplicative gain and fast readout architectures are also being developed... but as you said, cost starts playing the role.

cheers
shalin

On 10/6/07, Craig Brideau <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

So what's the catch?  Why do people still use PMTs vs. CCDs or EMCCDs?  Cost?

Craig

On 10/5/07, Kurt Thorn <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> In general CCDs are more sensitive that PMTs.  I can't speak to the
> sensitivities of the systems here, but a typical PMT has a quantum
> efficiency on the order of 10 - 25%, whereas good CCDs have QEs from 60
> - 90%.
>
> Kurt
>
> Craig Brideau wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Will this system have the same sort of sensitivity as a multi-anode
> > PMT system like the C1si?  This PARISS system seems to use a CCD
> > camera as its detector, which will not be as sensitive, if I
> > understand correctly?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> >
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Criag
> >> If spectral analysis is the endpoint you may want to consider the PARISS
> >> system from Lightform. ( http://www.lightforminc.com/ ) that can be
> >> placed on a widefield microscope.
> >> We are using the PARISS system to obtain spectral data --it has 1nm
> >> resolution and has a spectrum from 400-900nm. I find it to be a great
> >> asset in the laboratory,
> >> It is far more sensitive and accurate that any confocal system that I
> >> have seen.
> >>
> >> Contact Jeremy Lerner for details on his system.
> >> best wishes
> >> Bob
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>                                 LightForm, Inc.,
> >>                           601 Route 206, Suite 26-479
> >>                              Hillsborough NJ 08844
> >>                                Tel: (908)281 9098
> >>                         Email: [hidden email]
> >>
> >>
> >>
> >>
> >>
> >>
> >> .
> >>
> >> Robert M. Zucker, PhD
> >> U.S. Environmental Protection Agency
> >> Office of Research and Development
> >> National Health and Environmental Effects Research Laboratory.
> >> Telephone: 919-541-1585   Fax: 919-541-4017
> >> e-mail: [hidden email]
> >>
> >> Mail address: Reproductive Toxicology Division, MD 67
> >> 2525 E.NC Highway 54
> >> Research Triangle Park, North Carolina, 27711
> >>
> >> Shipping address: 2525 E.NC Highway 54
> >> Durham, NC, 27713
> >>
> >>
> >>
> >>
> >>              Craig Brideau
> >>              <craig.brideau@G
> >>              MAIL.COM>                                               To
> >>              Sent by:                 [hidden email]
> >>              Confocal                                                cc
> >>              Microscopy List
> >>              <CONFOCAL@LISTSE                                   Subject
> >>              RV.BUFFALO.EDU>          Re: Nikon C1si?
> >>
> >>
> >>              10/05/2007 05:48
> >>              PM
> >>
> >>
> >>               Please respond
> >>                     to
> >>                  Confocal
> >>              Microscopy List
> >>              <CONFOCAL@LISTSE
> >>              RV.BUFFALO.EDU>
> >>
> >>
> >>
> >>
> >>
> >>
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Thanks for your input!  As in everything, there's a trade-off.  Still,
> >> for us the spectral data will be worth it.
> >>
> >> Craig
> >>
> >> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> >>
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> Craig
> >>> The efficiency of detection is much less in the spectral mode due to
> >>>
> >> the
> >>
> >>> design of the multianode detector. In addition as Kurt Thorn said the
> >>> light is split into a number of different bandpass channels instead of
> >>> being detected by only one channel which also limits the detection in
> >>> each channel.
> >>> Nikon compensates for this decreased light by increasing the pixel
> >>>
> >> dwell
> >>
> >>> time. However photons are photons. With less photons you will get a
> >>> noisier image.
> >>> The spectral detection of any confocal system using a multianode
> >>> detector will not be as good as a PMT designed for good sensitivity
> >>>
> >> and
> >>
> >>> low light detection. It will produce nosier images but if there is
> >>> enough light you will be able to determine a valuable spectrum which
> >>>
> >> can
> >>
> >>> be used for scientific experiments and to manipulate images. Like
> >>> everything with confocal microscopy there is trade-offs and no perfect
> >>> system.
> >>> Best wishes.
> >>> Bob
> >>>
> >>> Robert M. Zucker, PhD
> >>> U.S. Environmental Protection Agency
> >>> Office of Research and Development
> >>> National Health and Environmental Effects Research Laboratory.
> >>> Telephone: 919-541-1585   Fax: 919-541-4017
> >>> e-mail: [hidden email]
> >>>
> >>> Mail address: Reproductive Toxicology Division, MD 67
> >>> 2525 E.NC Highway 54
> >>> Research Triangle Park, North Carolina, 27711
> >>>
> >>> Shipping address: 2525 E.NC Highway 54
> >>> Durham, NC, 27713
> >>>
> >>>
> >>>
> >>>
> >>>              Craig Brideau
> >>>              <craig.brideau@G
> >>>               MAIL.COM>
> >>>
> >> To
> >>
> >>>              Sent by:                 [hidden email]
> >>>              Confocal
> >>>
> >> cc
> >>
> >>>              Microscopy List
> >>>              <CONFOCAL@LISTSE
> >>>
> >> Subject
> >>
> >>>              RV.BUFFALO.EDU>          Re: Nikon C1si?
> >>>
> >>>
> >>>              10/04/2007 02:49
> >>>              PM
> >>>
> >>>
> >>>               Please respond
> >>>                     to
> >>>                  Confocal
> >>>              Microscopy List

> >>>              <CONFOCAL@LISTSE
> >>>              RV.BUFFALO.EDU>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>> Search the CONFOCAL archive at
> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> I was wondering if you saw this decreased throughput in spectral mode
> >>> only, or also in conventional detection mode?  The spectral mode works
> >>> with a multi-anode PMT, which has a lower quantum efficiency than a
> >>> conventional PMT.  From what I have seen of the design, it also has
> >>> the option to use a conventional PMT system with the device, and I am
> >>> wondering if you have also tried that mode of operation?
> >>>
> >>> Thanks,
> >>>
> >>> Craig
> >>>
> >>> On 10/4/07, Robert Zucker < [hidden email]> wrote:
> >>>
> >>>> One of the major limitations that we have seen with this unit and
> >>>>
> >> also
> >>
> >>>> with  the Zeiss meta 510 is the decreased light throughput . This
> >>>> creates images that are noisier than conventional confocal
> >>>>
> >>> microscopes.
> >>>
> >>>> In our hands it appears you will need to have a bright sample to
> >>>>
> >> make
> >>
> >>>> the spectral system work properly. ,
> >>>>
> >
> >
>
>
> --
> Kurt Thorn, PhD
> Director, Nikon Imaging Center
> University of California San Francisco
>
> UCSF MC 2140
> Genentech Hall Room S252
> 600 16th St.
> San Francisco, CA 94158-2517
>
> http://nic.ucsf.edu
> phone 415.514.9709
> fax   415.514.4300
>

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: electricsbm.blogspot.com
Mobile: +65 90694182

Shalin Mehta

Re: Nikon C1si?

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal sorry there was a typo...i meant... 'whereby one electron produced by photon is converted into a bunch of electrons'..

On 10/6/07, Shalin Mehta < [hidden email]> wrote:

Hi Craig,

CCDs can convert more photons into electrons in comparison to PMTs. But two major drawbacks are slow readout (due to semi-serial output) and no inherent multiplicative gain (whereby one electron produced by photon is converted into a bunch of photons..) A sufficiently powered PMT will give us a million or more electrons for each electron produced by photoelectric effect. It is easier for downstream electronics to handle large currents. EMCCDs can overcome the problem of multiplicative gain and fast readout architectures are also being developed... but as you said, cost starts playing the role.

cheers
shalin

On 10/6/07, Craig Brideau <[hidden email]> wrote:
Search the CONFOCAL archive at
<a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

So what's the catch?  Why do people still use PMTs vs. CCDs or EMCCDs?  Cost?

Craig

On 10/5/07, Kurt Thorn <[hidden email]> wrote:

> Search the CONFOCAL archive at
> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> In general CCDs are more sensitive that PMTs.  I can't speak to the
> sensitivities of the systems here, but a typical PMT has a quantum
> efficiency on the order of 10 - 25%, whereas good CCDs have QEs from 60
> - 90%.
>
> Kurt
>
> Craig Brideau wrote:
> > Search the CONFOCAL archive at
> > <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Will this system have the same sort of sensitivity as a multi-anode
> > PMT system like the C1si?  This PARISS system seems to use a CCD
> > camera as its detector, which will not be as sensitive, if I
> > understand correctly?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> >
> >> Search the CONFOCAL archive at
> >> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Criag
> >> If spectral analysis is the endpoint you may want to consider the PARISS
> >> system from Lightform. ( <a href="http://www.lightforminc.com/" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://www.lightforminc.com/ ) that can be
> >> placed on a widefield microscope.
> >> We are using the PARISS system to obtain spectral data --it has 1nm
> >> resolution and has a spectrum from 400-900nm. I find it to be a great
> >> asset in the laboratory,
> >> It is far more sensitive and accurate that any confocal system that I
> >> have seen.
> >>
> >> Contact Jeremy Lerner for details on his system.
> >> best wishes
> >> Bob
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>                                 LightForm, Inc.,
> >>                           601 Route 206, Suite 26-479
> >>                              Hillsborough NJ 08844
> >>                                Tel: (908)281 9098
> >>                         Email: [hidden email]
> >>
> >>
> >>
> >>
> >>
> >>
> >> .
> >>
> >> Robert M. Zucker, PhD
> >> U.S. Environmental Protection Agency
> >> Office of Research and Development
> >> National Health and Environmental Effects Research Laboratory.
> >> Telephone: 919-541-1585   Fax: 919-541-4017
> >> e-mail: [hidden email]
> >>
> >> Mail address: Reproductive Toxicology Division, MD 67
> >> 2525 <a href="http://E.NC" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> E.NC Highway 54
> >> Research Triangle Park, North Carolina, 27711
> >>
> >> Shipping address: 2525 <a href="http://E.NC" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> E.NC Highway 54
> >> Durham, NC, 27713
> >>
> >>
> >>
> >>
> >>              Craig Brideau
> >>              <craig.brideau@G
> >>              <a href="http://MAIL.COM" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> MAIL.COM>                                               To
> >>              Sent by:                 [hidden email]
> >>              Confocal                                                cc
> >>              Microscopy List
> >>              <CONFOCAL@LISTSE                                   Subject
> >>              <a href="http://RV.BUFFALO.EDU" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> RV.BUFFALO.EDU>          Re: Nikon C1si?
> >>
> >>
> >>              10/05/2007 05:48
> >>              PM
> >>
> >>
> >>               Please respond
> >>                     to
> >>                  Confocal
> >>              Microscopy List
> >>              <CONFOCAL@LISTSE
> >>              <a href="http://RV.BUFFALO.EDU" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> RV.BUFFALO.EDU>
> >>
> >>
> >>
> >>
> >>
> >>
> >> Search the CONFOCAL archive at
> >> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Thanks for your input!  As in everything, there's a trade-off.  Still,
> >> for us the spectral data will be worth it.
> >>
> >> Craig
> >>
> >> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> >>
> >>> Search the CONFOCAL archive at
> >>> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>

> >>> Craig
> >>> The efficiency of detection is much less in the spectral mode due to
> >>>
> >> the
> >>
> >>> design of the multianode detector. In addition as Kurt Thorn said the
> >>> light is split into a number of different bandpass channels instead of
> >>> being detected by only one channel which also limits the detection in
> >>> each channel.
> >>> Nikon compensates for this decreased light by increasing the pixel
> >>>
> >> dwell
> >>
> >>> time. However photons are photons. With less photons you will get a
> >>> noisier image.
> >>> The spectral detection of any confocal system using a multianode
> >>> detector will not be as good as a PMT designed for good sensitivity
> >>>
> >> and
> >>
> >>> low light detection. It will produce nosier images but if there is
> >>> enough light you will be able to determine a valuable spectrum which
> >>>
> >> can
> >>
> >>> be used for scientific experiments and to manipulate images. Like
> >>> everything with confocal microscopy there is trade-offs and no perfect
> >>> system.
> >>> Best wishes.
> >>> Bob
> >>>
> >>> Robert M. Zucker, PhD
> >>> U.S. Environmental Protection Agency
> >>> Office of Research and Development
> >>> National Health and Environmental Effects Research Laboratory.
> >>> Telephone: 919-541-1585   Fax: 919-541-4017
> >>> e-mail: [hidden email]
> >>>
> >>> Mail address: Reproductive Toxicology Division, MD 67
> >>> 2525 <a href="http://E.NC" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> E.NC Highway 54
> >>> Research Triangle Park, North Carolina, 27711
> >>>
> >>> Shipping address: 2525 <a href="http://E.NC" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> E.NC Highway 54
> >>> Durham, NC, 27713
> >>>
> >>>
> >>>
> >>>
> >>>              Craig Brideau
> >>>              <craig.brideau@G
> >>>               <a href="http://MAIL.COM" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">MAIL.COM>
> >>>
> >> To
> >>
> >>>              Sent by:                 [hidden email]
> >>>              Confocal
> >>>
> >> cc
> >>
> >>>              Microscopy List
> >>>              <CONFOCAL@LISTSE
> >>>
> >> Subject
> >>
> >>>              <a href="http://RV.BUFFALO.EDU" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">RV.BUFFALO.EDU>          Re: Nikon C1si?
> >>>
> >>>
> >>>              10/04/2007 02:49
> >>>              PM
> >>>
> >>>
> >>>               Please respond
> >>>                     to
> >>>                  Confocal
> >>>              Microscopy List

> >>>              <CONFOCAL@LISTSE
> >>>              <a href="http://RV.BUFFALO.EDU" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> RV.BUFFALO.EDU>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>>
> >>> Search the CONFOCAL archive at
> >>> <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>>
> >>> I was wondering if you saw this decreased throughput in spectral mode
> >>> only, or also in conventional detection mode?  The spectral mode works
> >>> with a multi-anode PMT, which has a lower quantum efficiency than a
> >>> conventional PMT.  From what I have seen of the design, it also has
> >>> the option to use a conventional PMT system with the device, and I am
> >>> wondering if you have also tried that mode of operation?
> >>>
> >>> Thanks,
> >>>
> >>> Craig
> >>>
> >>> On 10/4/07, Robert Zucker < [hidden email]> wrote:
> >>>
> >>>> One of the major limitations that we have seen with this unit and
> >>>>
> >> also
> >>
> >>>> with  the Zeiss meta 510 is the decreased light throughput . This
> >>>> creates images that are noisier than conventional confocal
> >>>>
> >>> microscopes.
> >>>
> >>>> In our hands it appears you will need to have a bright sample to
> >>>>
> >> make
> >>
> >>>> the spectral system work properly. ,
> >>>>
> >
> >
>
>
> --
> Kurt Thorn, PhD
> Director, Nikon Imaging Center
> University of California San Francisco
>
> UCSF MC 2140
> Genentech Hall Room S252
> 600 16th St.
> San Francisco, CA 94158-2517
>
> <a href="http://nic.ucsf.edu" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://nic.ucsf.edu
> phone 415.514.9709
> fax   415.514.4300
>

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: <a href="http://electricsbm.blogspot.com" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> electricsbm.blogspot.com
Mobile: +65 90694182

James Pawley

Re: Nikon C1si?

In reply to this post by Guy Cox

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi all,

About which is a better photodetector for spectral confocal, one
based on PMTs or one based on CCDs:

True: the CCD can have a QE of 90% (though most are closer to 50%)
while the EFFECTIVE QE of a single-channel
"good-but-not-so-super-good-that-it-needs-cooling" PMT is more likely
2-10% (depending on wavelength, and I am being generous, while
including all the factors that reduce the listed QE, such as lost
charge and multiplicative noise.)

But this is only PART of the problem. The other main factor is the
read noise. THe read noise of the PMT is essentially zero, but unless
one reads out the CCD very slowly (less than 0.1 MHz, not really
possible is you are to record an entire spectrum at each pixel) then
CCD read noise will be at least +/-10 electrons (and may be closer to
+/- 20e).

This means that any signal less than 20 electrons/CCD pixel, will be
lost in the noise. Assuming that the CCD-QE is 10x higher than the
PMT-QE, then this 20 electron signal in the CCD would produce only 2
electrons in the PMT. The point is that even before you split the
signal into 32 channels (or even the 4 or 5 channels that cover the
bandwidth of a the emission spectrum of most dyes) the signal getting
through the pinhole is in the tens of photons from a bright pixel.
Consequently, it is often 0 photons from an unstained area of the
cell. CCDs are very poor at measuring zero light.

So until the manufacturers start making linear EM-CCD with few enough
channels (i.e., 10-30) to work in spectral confocal, PMT arrays will
still find a useful application.

Those who find that ordinary scientific CCDs are useful in
disk-scanning confocal or spectral imaging are those who have a LOT
of signal. Probably 100x more than would be needed with an EM-CCD.

This could be either because they are using more excitation light or
because there are more dye molecules in each interrogated volume. In
any case, as phototoxicity is proportional to EXCITATIONS rather than
to incident light, the emission of this much signal is likely to be
unpleasant to the cell.

Cheers,

Jim P.
--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-262-9083
250 N. Mills St., Madison, WI, 53706 [hidden email]
"A scientist is not one who can answer questions but one who can
question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39

George McNamara

Re: Nikon C1si? -> PARISS can be used as a slit confocal for optical slicing

In reply to this post by Craig Brideau

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Craig,

The Lightform PARISS has a detection slit in front of the spectral dispersion element and the CCD. This makes it half-confocal. If a matching slit is placed in the epi-illumination field aperture plane (or a conjugate plane) it is a slit confocal (aka line scan confocal). The result is an X*Spectral image. If the microscope has a motorized stage, the Y-axis can be added = push broom confocal imaging spectrometer. The performance is such that the emission filter can be removed from the epi-illumination filter cube when used with an Hg lamp (the bright Hg line or lines are one nanometer or so side = 1 or a few pixels, so do not bleed into the emission areas and can be used to quantify the excitation power). The PARISS can also be used for brightfield images.

You can contact Jeremy Lerner at http://www.lightforminc.com/ for additional details about the PARISS.

With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point scanning confocal microscope detects a maximum of about 8 photons per pixel per scan. This is a nice match to a PMT (or APD for far red and infrared photons), not so good for a CCD (see Jim's post about noise). On the other hand, a CCD is an area detector (or line * spectrum detector for the PARISS), so the CCD is a great way to acquire 2D images fast. A CCD or EMCCD system can be used to image a thin optical section, quickly, with judicious use of excitation light, as in TIRF, fast multipoint multiphoton scanners such as the TriMScope (if it can be made to work reliably) or Scherer's stochastic scanner ( http://www.opticsexpress.org/abstract.cfm?id=89328), or hopefully by widefield multiphoton excitation (Brooker 2007 US patent 7,170,675 ), or by multiplane acquisition followed by digital deconvolution. If you can buy or borrow 3 EMCCD's like Ian Parker, you can acquire multiple focal planes simultaneously, see PubMed 17716727, also similar rig by Prabhat, PubMed 17384151.

Back to 32-channel PMT confocal microscopes: the Zeiss META detector includes blockers for the major laser lines. As far as I am aware, they are in the light path all the time (the 488 nm blocker may be responsible for zigzag artifacts in CFP emission spectra, though bad PMT channels could also be responsible). Nikon may include similar blockers in the si. The Zeiss LSM510 light path can be configured to use a microscope filter cube. With the right cube and a mirror slide, you can acquire the spectrum of the Hg lamp using the META, or you could buy a Lightform MIDL like Bob Zucker's and just lay it on the microscope. The META spectrum of the MIDL lamp is not even close to the calibration curve that comes with the MIDL. Bob and Jeremy have published the close match of the PARISS data for the MIDL.

Enjoy,

George

At 07:26 PM 10/5/2007, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks! We are interested in co-localization, for which we sometimes
need the third dimension. Mainly we want to do spectral unmixing for
multiple dyes and colocalization, so we really do need optical
slicing!

Thanks,

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Craig
> you are correct.
> Currently the PARISS only detects X-Y--not depth.
> however, most data that I have seen from confocal spectral systems are
> only a single XY slice. they usually are not used to do a 3D
> reconstruction in spectral mode.
> bob
> .
>
>
> Robert M. Zucker, PhD
> U.S. Environmental Protection Agency
> Office of Research and Development
> National Health and Environmental Effects Research Laboratory.
> Telephone: 919-541-1585   Fax: 919-541-4017
> e-mail: [hidden email]
>
> Mail address: Reproductive Toxicology Division, MD 67
> 2525 E.NC Highway 54
> Research Triangle Park, North Carolina, 27711
>
> Shipping address: 2525 E.NC Highway 54
> Durham, NC, 27713
>
>
>
>
>              Craig Brideau
>              <craig.brideau@G
>              MAIL.COM>                                               To
>              Sent by:                 [hidden email]
>              Confocal                                                cc
>              Microscopy List
>              <CONFOCAL@LISTSE                                   Subject
>              RV.BUFFALO.EDU>          Re: Nikon C1si?
>
>
>              10/05/2007 06:44
>              PM
>
>
>               Please respond
>                     to
>                  Confocal
>              Microscopy List
>              <CONFOCAL@LISTSE
>              RV.BUFFALO.EDU>
>
>
>
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> It doesn't seem to deal with depth though? It only detects in X-Y, so
> can it generate optical slices?
>
> Craig
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> more
> > sensitive and accurate than any confocal microscope.
> > You can detect things in that system that can not be observed with a
> > confocal spectral system. It is a great system in my opinion.
> > Bob
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585   Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> >              Craig Brideau
> >              <craig.brideau@G
> >              MAIL.COM>
> To
> >              Sent by:                 [hidden email]
> >              Confocal
> cc
> >              Microscopy List
> >              <CONFOCAL@LISTSE
> Subject
> >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> >
> >
> >              10/05/2007 06:19
> >              PM
> >
> >
> >               Please respond
> >                     to
> >                  Confocal
> >              Microscopy List
> >              <CONFOCAL@LISTSE
> >              RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Will this system have the same sort of sensitivity as a multi-anode
> > PMT system like the C1si? This PARISS system seems to use a CCD
> > camera as its detector, which will not be as sensitive, if I
> > understand correctly?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Criag
> > > If spectral analysis is the endpoint you may want to consider the
> > PARISS
> > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > placed on a widefield microscope.
> > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > resolution and has a spectrum from 400-900nm. I find it to be a
> great
> > > asset in the laboratory,
> > > It is far more sensitive and accurate that any confocal system that
> I
> > > have seen.
> > >
> > > Contact Jeremy Lerner for details on his system.
> > > best wishes
> > > Bob
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >
> > >                                 LightForm, Inc.,
> > >                           601 Route 206, Suite 26-479
> > >                              Hillsborough NJ 08844
> > >                                Tel: (908)281 9098
> > >                         Email: [hidden email]
> > >
> > >
> > >
> > >
> > >
> > >
> > > .
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585   Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > >              Craig Brideau
> > >              <craig.brideau@G
> > >              MAIL.COM>
> > To
> > >              Sent by:                 [hidden email]
> > >              Confocal
> > cc
> > >              Microscopy List
> > >              <CONFOCAL@LISTSE
> > Subject
> > >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> > >
> > >
> > >              10/05/2007 05:48
> > >              PM
> > >
> > >
> > >               Please respond
> > >                     to
> > >                  Confocal
> > >              Microscopy List
> > >              <CONFOCAL@LISTSE
> > >              RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Thanks for your input! As in everything, there's a trade-off.
> Still,
> > > for us the spectral data will be worth it.
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > Search the CONFOCAL archive at
> > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Craig
> > > > The efficiency of detection is much less in the spectral mode due
> to
> > > the
> > > > design of the multianode detector. In addition as Kurt Thorn said
> > the
> > > > light is split into a number of different bandpass channels
> instead
> > of
> > > > being detected by only one channel which also limits the detection
> > in
> > > > each channel.
> > > > Nikon compensates for this decreased light by increasing the pixel
> > > dwell
> > > > time. However photons are photons. With less photons you will get
> a
> > > > noisier image.
> > > > The spectral detection of any confocal system using a multianode
> > > > detector will not be as good as a PMT designed for good
> sensitivity
> > > and
> > > > low light detection. It will produce nosier images but if there is
> > > > enough light you will be able to determine a valuable spectrum
> which
> > > can
> > > > be used for scientific experiments and to manipulate images. Like
> > > > everything with confocal microscopy there is trade-offs and no
> > perfect
> > > > system.
> > > > Best wishes.
> > > > Bob
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585   Fax: 919-541-4017
> > > > e-mail: [hidden email]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > >              Craig Brideau
> > > >              <craig.brideau@G
> > > >              MAIL.COM>
> > > To
> > > >              Sent by:
> [hidden email]
> > > >              Confocal
> > > cc
> > > >              Microscopy List
> > > >              <CONFOCAL@LISTSE
> > > Subject
> > > >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> > > >
> > > >
> > > >              10/04/2007 02:49
> > > >              PM
> > > >
> > > >
> > > >               Please respond
> > > >                     to
> > > >                  Confocal
> > > >              Microscopy List
> > > >              <CONFOCAL@LISTSE
> > > >              RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > I was wondering if you saw this decreased throughput in spectral
> > mode
> > > > only, or also in conventional detection mode? The spectral mode
> > works
> > > > with a multi-anode PMT, which has a lower quantum efficiency than
> a
> > > > conventional PMT. From what I have seen of the design, it also
> has
> > > > the option to use a conventional PMT system with the device, and I
> > am
> > > > wondering if you have also tried that mode of operation?
> > > >
> > > > Thanks,
> > > >
> > > > Craig
> > > >
> > > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > > One of the major limitations that we have seen with this unit
> and
> > > also
> > > > > with the Zeiss meta 510 is the decreased light throughput .
> This
> > > > > creates images that are noisier than conventional confocal
> > > > microscopes.
> > > > > In our hands it appears you will need to have a bright sample to
> > > make
> > > > > the spectral system work properly. ,
> > > >
> > >
> >
>

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp (see Analytical Imaging Core Facility)

Craig Brideau

Re: Nikon C1si? -> PARISS can be used as a slit confocal for optical slicing

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Hi Craig,
>
> The Lightform PARISS has a detection slit in front of the spectral
> dispersion element and the CCD. This makes it half-confocal. If a matching
> slit is placed in the epi-illumination field aperture plane (or a conjugate
> plane) it is a slit confocal (aka line scan confocal). The result is an
> X*Spectral image. If the microscope has a motorized stage, the Y-axis can be
> added = push broom confocal imaging spectrometer. The performance is such
> that the emission filter can be removed from the epi-illumination filter
> cube when used with an Hg lamp (the bright Hg line or lines are one
> nanometer or so side = 1 or a few pixels, so do not bleed into the emission
> areas and can be used to quantify the excitation power). The PARISS can also
> be used for brightfield images.
>
> You can contact Jeremy Lerner at http://www.lightforminc.com/ for
> additional details about the PARISS.
>
>
> With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point
> scanning confocal microscope detects a maximum of about 8 photons per pixel
> per scan. This is a nice match to a PMT (or APD for far red and infrared
> photons), not so good for a CCD (see Jim's post about noise). On the other
> hand, a CCD is an area detector (or line * spectrum detector for the
> PARISS), so the CCD is a great way to acquire 2D images fast. A CCD or EMCCD
> system can be used to image a thin optical section, quickly, with judicious
> use of excitation light, as in TIRF, fast multipoint multiphoton scanners
> such as the TriMScope (if it can be made to work reliably) or Scherer's
> stochastic scanner (
> http://www.opticsexpress.org/abstract.cfm?id=89328), or
> hopefully by widefield multiphoton excitation (Brooker 2007 US patent
> 7,170,675 ), or by multiplane acquisition followed by digital deconvolution.
> If you can buy or borrow 3 EMCCD's like Ian Parker, you can acquire multiple
> focal planes simultaneously, see PubMed 17716727, also similar rig by
> Prabhat, PubMed 17384151.
>
> Back to 32-channel PMT confocal microscopes: the Zeiss META detector
> includes blockers for the major laser lines. As far as I am aware, they are
> in the light path all the time (the 488 nm blocker may be responsible for
> zigzag artifacts in CFP emission spectra, though bad PMT channels could also
> be responsible). Nikon may include similar blockers in the si. The Zeiss
> LSM510 light path can be configured to use a microscope filter cube. With
> the right cube and a mirror slide, you can acquire the spectrum of the Hg
> lamp using the META, or you could buy a Lightform MIDL like Bob Zucker's and
> just lay it on the microscope. The META spectrum of the MIDL lamp is not
> even close to the calibration curve that comes with the MIDL. Bob and Jeremy
> have published the close match of the PARISS data for the MIDL.
>
>
> Enjoy,
>
>
> George
>
>
>
> At 07:26 PM 10/5/2007, you wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Thanks! We are interested in co-localization, for which we sometimes
> need the third dimension. Mainly we want to do spectral unmixing for
> multiple dyes and colocalization, so we really do need optical
> slicing!
>
> Thanks,
>
> Craig
>
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Craig
> > you are correct.
> > Currently the PARISS only detects X-Y--not depth.
> > however, most data that I have seen from confocal spectral systems are
> > only a single XY slice. they usually are not used to do a 3D
> > reconstruction in spectral mode.
> > bob
> > .
> >
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585 Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> > Craig Brideau
> > <craig.brideau@G
> > MAIL.COM>
> To
> > Sent by: [hidden email]
> > Confocal
> cc
> > Microscopy List
> > <CONFOCAL@LISTSE
> Subject
> > RV.BUFFALO.EDU> Re: Nikon C1si?
> >
> >
> > 10/05/2007 06:44
> > PM
> >
> >
> > Please respond
> > to
> > Confocal
> > Microscopy List
> > <CONFOCAL@LISTSE
> > RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > It doesn't seem to deal with depth though? It only detects in X-Y, so
> > can it generate optical slices?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> > more
> > > sensitive and accurate than any confocal microscope.
> > > You can detect things in that system that can not be observed with a
> > > confocal spectral system. It is a great system in my opinion.
> > > Bob
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by: [hidden email]
> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/05/2007 06:19
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Will this system have the same sort of sensitivity as a multi-anode
> > > PMT system like the C1si? This PARISS system seems to use a CCD
> > > camera as its detector, which will not be as sensitive, if I
> > > understand correctly?
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Criag
> > > > If spectral analysis is the endpoint you may want to consider the
> > > PARISS
> > > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > > placed on a widefield microscope.
> > > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > > resolution and has a spectrum from 400-900nm. I find it to be a
> > great
> > > > asset in the laboratory,
> > > > It is far more sensitive and accurate that any confocal system that
> > I
> > > > have seen.
> > > >
> > > > Contact Jeremy Lerner for details on his system.
> > > > best wishes
> > > > Bob
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > LightForm, Inc.,
> > > > 601 Route 206, Suite 26-479
> > > > Hillsborough NJ 08844
> > > > Tel: (908)281 9098
> > > > Email: [hidden email]
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > .
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > e-mail: [hidden email]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > > Craig Brideau
> > > > <craig.brideau@G
> > > > MAIL.COM>
> > > To
> > > > Sent by: [hidden email]
> > > > Confocal
> > > cc
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > Subject
> > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > >
> > > >
> > > > 10/05/2007 05:48
> > > > PM
> > > >
> > > >
> > > > Please respond
> > > > to
> > > > Confocal
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > > RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Thanks for your input! As in everything, there's a trade-off.
> > Still,
> > > > for us the spectral data will be worth it.
> > > >
> > > > Craig
> > > >
> > > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > Craig
> > > > > The efficiency of detection is much less in the spectral mode due
> > to
> > > > the
> > > > > design of the multianode detector. In addition as Kurt Thorn said
> > > the
> > > > > light is split into a number of different bandpass channels
> > instead
> > > of
> > > > > being detected by only one channel which also limits the detection
> > > in
> > > > > each channel.
> > > > > Nikon compensates for this decreased light by increasing the pixel
> > > > dwell
> > > > > time. However photons are photons. With less photons you will get
> > a
> > > > > noisier image.
> > > > > The spectral detection of any confocal system using a multianode
> > > > > detector will not be as good as a PMT designed for good
> > sensitivity
> > > > and
> > > > > low light detection. It will produce nosier images but if there is
> > > > > enough light you will be able to determine a valuable spectrum
> > which
> > > > can
> > > > > be used for scientific experiments and to manipulate images. Like
> > > > > everything with confocal microscopy there is trade-offs and no
> > > perfect
> > > > > system.
> > > > > Best wishes.
> > > > > Bob
> > > > >
> > > > > Robert M. Zucker, PhD
> > > > > U.S. Environmental Protection Agency
> > > > > Office of Research and Development
> > > > > National Health and Environmental Effects Research Laboratory.
> > > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > > e-mail: [hidden email]
> > > > >
> > > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > > 2525 E.NC Highway 54
> > > > > Research Triangle Park, North Carolina, 27711
> > > > >
> > > > > Shipping address: 2525 E.NC Highway 54
> > > > > Durham, NC, 27713
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Craig Brideau
> > > > > <craig.brideau@G
> > > > > MAIL.COM>
> > > > To
> > > > > Sent by:
> > [hidden email]
> > > > > Confocal
> > > > cc
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > Subject
> > > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > > >
> > > > >
> > > > > 10/04/2007 02:49
> > > > > PM
> > > > >
> > > > >
> > > > > Please respond
> > > > > to
> > > > > Confocal
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > > RV.BUFFALO.EDU>
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > I was wondering if you saw this decreased throughput in spectral
> > > mode
> > > > > only, or also in conventional detection mode? The spectral mode
> > > works
> > > > > with a multi-anode PMT, which has a lower quantum efficiency than
> > a
> > > > > conventional PMT. From what I have seen of the design, it also
> > has
> > > > > the option to use a conventional PMT system with the device, and I
> > > am
> > > > > wondering if you have also tried that mode of operation?
> > > > >
> > > > > Thanks,
> > > > >
> > > > > Craig
> > > > >
> > > > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > > > One of the major limitations that we have seen with this unit
> > and
> > > > also
> > > > > > with the Zeiss meta 510 is the decreased light throughput .
> > This
> > > > > > creates images that are noisier than conventional confocal
> > > > > microscopes.
> > > > > > In our hands it appears you will need to have a bright sample to
> > > > make
> > > > > > the spectral system work properly. ,
> > > > >
> > > >
> > >
> >
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp
> (see Analytical Imaging Core Facility)
>
>
>

Karl Garsha

Re: Nikon C1si?

In reply to this post by Craig Brideau

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello all,

There are several points to consider on the issue of sensitivity and the tradeoffs between signal to noise ratio and optical sectioning capability with regard to different genres of spectral imaging instrument, different approaches have different strengths and weaknesses.

Sensitivity is a complex term. I think it is important to discern between quantum efficiency and amplification; detectors with high quantum efficiency get a high percentage of the available signal from a given optics train. Detectors with high amplification take the portion of available signal that is detected and amplify it to a much higher signature. As a general rule, scientific grade CCD detectors have higher quantum efficiency than PMT's. A typical front-illuminated, microlensed CCD will have a QE on the order of 60-70%, a back-thinned CCD may have a QE on the order of 90-95%, and a PMT's generally have a lower effective QE as Jim pointed out. However, conventional CCD's don't amplify the signal prior to readout, PMT's do (by as much as factor of a million times). EMCCD cameras are generally backthinned for high quantum efficiency and also amplify the signal prior to readout (this provides a benefit to the signal to noise ratio because the readout noise is not amplified with the signal), although the signal is amplified to a lesser extent than with PMT's. It is important to consider the sources of noise in addition to QE when discussing detector technologies, the signal-to-noise ratio is generally the concept people are trying to allude to when discussing sensitivity. Noise can arise from the physical nature of photon counting statistics (shot noise), the readout electronics (read noise), the signal amplification process (excess noise factor, spurious charge) and dark current. Jim Pawley's 'Handbook of Biological Confocal Microscopy, 3rd edition' discusses such concepts in depth and is an excellent resource. The proportional impact of different noise sources and the signal-to-noise ratio that can be achieved by a given detector technology depends on the technology itself convolved with the context in which it is used.

Spectral imaging is a systems level challenge, in contrast to a problem that can be solved with a detector strategy alone. A salient point to consider is the strategy with which multidimensional spectral data is acquired. As has already been observed, point scanning instruments offer exquisite optical sectioning capability with the disadvantage of fewer photons contributing to the overall signal/pixel for a given rate of image acquisition, with a sample of given quantum yield, at a given resolution and a given illumination dosage. For truly unambiguous determination of the 3-D origin of a spectral 'super-pixel', it could be argued that non-linear (multiphoton) excitation provides significant advantages. Available CCD based instruments collect information from many pixels in parallel, so the integration time for each pixel at a given exposure is longer and this provides the opportunity to collect more photons at a given exposure time. Because CCD's are two-dimensional arrays of detectors, many pixels of spatial information can be collected in parallel along one dimension, while the entire spectra at each of these pixels is dispersed across (and collected) in the second dimension of the CCD chip...this is the paradigm that gives rise to slit based, widefield illumination spectral detection systems. Such instruments may collect an entire single row of pixels of spatial information per exposure; in order to collect information from the entire field of view, either the sample or the slit aperture is moved to an adjacent position and another exposure is acquired. This process is repeated until the entire dataset of 2 spatial dimensions with spectral information at each pixel is acquired. But because the widefield system is acquiring spatial data one row of pixels at a time as opposed to one pixel at a time (as in the point scanning instrument), the amount of time each pixel is permitted to integrate photons for a given rate of acquisition is longer by a factor of how ever many pixels there are in a row (assuming the same number of pixels/row between instruments).

Yet another issue to consider is photobleaching and the possibility of phototoxicity (where living samples are concerned). This is a complex subject as well but it can be generalized that point scanning instruments only illuminate single pixels as they are being imaged (albiet at a high intensity) while a widefield instrument may illuminate the entire field of view during the entire time the data-set is being acquired. In sensitive samples, this may mean that the entire field is photo-bleaching while pixels are only being imaged one row at a time. One solution to this approach is to provide a matching illumination slit in a plane confocal to the detection aperture on a slit-based, widefield illumination spectral imaging system. This restricts photobleaching to the row of pixels being acquired and provides some rejection of extraneous light from out-of-focus planes; there is at least one widefield system on the market that takes this approach and couples it to a back-thinned EMCCD detector.

There are several other approaches to the challenge of spectral imaging as well: LCD filters that change color and permit collection of 2-D images at different wavelengths in serial, point-scanning instruments that collect spectral data in serial by changing the detection range after each image in turn, frequency space fourier-transform based spectral imaging devices, and, in the basic research arena, a technology that uses a holographic grating to disperse entire 2-D images across a large CCD to permit parallel collection of spectral and 2-D spatial data. Presently the point scanning confocal and line-scanning widefield approaches tend to be the most relevant to fluorescence imaging applications in the biological sciences, the best tool for the job depends on the relative importance of a number of features/capabilities and each approach has it's merits.

Sincere Regards,
Karl

MAG Signature

Karl Garsha
Head Application Scientist
Microimaging Applications Group (MAG)
3440 East Britannia Drive
Tucson, AZ 85629
tel 520.889.9933
direct 520.547.2704
fax 520.573.1944

MAG Biosystems | Photometrics | QImaging | Media Cybernetics | Gatan
www.MAGbiosystems.com www.photomet.com www.qimaging.com www.mediacy.com www.gatan.com

The Microimaging Applications Group comprises five imaging leaders: Photometrics, Gatan, Media Cybernetics,
QImaging, and MAG Biosystems. These technology partners work independently as well as in synergy to offer an
unparalleled range of solutions for microimaging applications.

This message and any attachments are solely for the use of intended recipients. They may contain privileged and/or confidential information.
If you are not the intended recipient, you are hereby notified that you received this email in error, and that any review, dissemination, distribution
or copying of this email and any attachment is strictly prohibited. If you receive this email in error please contact the sender and delete the
message and any attachments associated therewith from your computer. Your cooperation in this matter is appreciated.

Craig Brideau wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

So what's the catch?  Why do people still use PMTs vs. CCDs or EMCCDs?  Cost?

Craig


On 10/5/07, Kurt Thorn [hidden email] wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In general CCDs are more sensitive that PMTs.  I can't speak to the
sensitivities of the systems here, but a typical PMT has a quantum
efficiency on the order of 10 - 25%, whereas good CCDs have QEs from 60
- 90%.

Kurt

Craig Brideau wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Will this system have the same sort of sensitivity as a multi-anode
PMT system like the C1si?  This PARISS system seems to use a CCD
camera as its detector, which will not be as sensitive, if I
understand correctly?

Craig

On 10/5/07, Robert Zucker [hidden email] wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Criag
If spectral analysis is the endpoint you may want to consider the PARISS
system from Lightform. ( http://www.lightforminc.com/) that can be
placed on a widefield microscope.
We are using the PARISS system to obtain spectral data --it has 1nm
resolution and has a spectrum from 400-900nm. I find it to be a great
asset in the laboratory,
It is far more sensitive and accurate that any confocal system that I
have seen.

Contact Jeremy Lerner for details on his system.
best wishes
Bob








                                LightForm, Inc.,
                          601 Route 206, Suite 26-479
                             Hillsborough NJ 08844
                               Tel: (908)281 9098
                        Email: [hidden email]






.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585   Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713




             Craig Brideau
             [hidden email]                                               To
             Sent by:                 [hidden email]
             Confocal                                                cc
             Microscopy List
             [hidden email]          Re: Nikon C1si?


             10/05/2007 05:48
             PM


              Please respond
                    to
                 Confocal
             Microscopy List
             [hidden email]






Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for your input!  As in everything, there's a trade-off.  Still,
for us the spectral data will be worth it.

Craig

On 10/5/07, Robert Zucker [hidden email] wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
The efficiency of detection is much less in the spectral mode due to

the

design of the multianode detector. In addition as Kurt Thorn said the
light is split into a number of different bandpass channels instead of
being detected by only one channel which also limits the detection in
each channel.
Nikon compensates for this decreased light by increasing the pixel

dwell

time. However photons are photons. With less photons you will get a
noisier image.
The spectral detection of any confocal system using a multianode
detector will not be as good as a PMT designed for good sensitivity

and

low light detection. It will produce nosier images but if there is
enough light you will be able to determine a valuable spectrum which

can

be used for scientific experiments and to manipulate images. Like
everything with confocal microscopy there is trade-offs and no perfect
system.
Best wishes.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585   Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713




             Craig Brideau
             [hidden email]

To

             Sent by:                 [hidden email]
             Confocal

cc

             Microscopy List
             <CONFOCAL@LISTSE

Subject

             RV.BUFFALO.EDU>          Re: Nikon C1si?


             10/04/2007 02:49
             PM


              Please respond
                    to
                 Confocal
             Microscopy List
             [hidden email]






Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I was wondering if you saw this decreased throughput in spectral mode
only, or also in conventional detection mode?  The spectral mode works
with a multi-anode PMT, which has a lower quantum efficiency than a
conventional PMT.  From what I have seen of the design, it also has
the option to use a conventional PMT system with the device, and I am
wondering if you have also tried that mode of operation?

Thanks,

Craig

On 10/4/07, Robert Zucker [hidden email] wrote:

One of the major limitations that we have seen with this unit and

also

with  the Zeiss meta 510 is the decreased light throughput . This
creates images that are noisier than conventional confocal

microscopes.

In our hands it appears you will need to have a bright sample to

make

the spectral system work properly. ,

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone 415.514.9709
fax   415.514.4300

JOEL B. SHEFFIELD

CCD vs PMT - Sensitivity vs Signal/Noise was Nikon C1si?

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I hope the list doesn't mind. I have been following this fascinating
thread for some time, but I realize that the discussion has strayed
from the original topic. I am taking the liberty of re-posting
Karl's offering mostly so that others who are searching the archives
will find the discussion.

Joel

-------------- Original message ---------------
Date sent: Sun, 7 Oct 2007 14:33:58 -0700
Send reply to: [hidden email]
From: Karl Garsha <[hidden email]>
Organization: MAG Biosystems
Subject: Re: Nikon C1si?
To: [hidden email]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-
bin/wa?S1=confocal
Hello all,

There are several points to consider on the issue of sensitivity and
the tradeoffs between signal to noise ratio and optical sectioning
capability with regard to different genres of spectral imaging
instrument, different approaches have different strengths and
weaknesses.

Sensitivity is a complex term. I think it is important to discern
between quantum efficiency and amplification; detectors with high
quantum efficiency get a high percentage of the available signal from
a given optics train. Detectors with high amplification take the
portion of available signal that is detected and amplify it to a much
higher signature. As a general rule, scientific grade CCD detectors
have higher quantum efficiency than PMT's. A typical front-
illuminated, microlensed CCD will have a QE on the order of 60-70%, a
back-thinned CCD may have a QE on the order of 90-95%, and a PMT's
generally have a lower effective QE as Jim pointed out. However,
conventional CCD's don't amplify the signal prior to readout, PMT's
do (by as much as factor of a million times). EMCCD cameras are
generally backthinned for high quantum efficiency and also amplify
the signal prior to readout (this provides a benefit to the signal to
noise ratio because the readout noise is not amplified with the
signal), although the signal is amplified to a lesser extent than
with PMT's. It is important to consider the sources of noise in
addition to QE when discussing detector technologies, the signal-to-
noise ratio is generally the concept people are trying to allude to
when discussing sensitivity. Noise can arise from the physical nature
of photon counting statistics (shot noise), the readout electronics
(read noise), the signal amplification process (excess noise factor,
spurious charge) and dark current. Jim Pawley's 'Handbook of
Biological Confocal Microscopy, 3rd edition' discusses such concepts
in depth and is an excellent resource. The proportional impact of
different noise sources and the signal-to-noise ratio that can be
achieved by a given detector technology depends on the technology
itself convolved with the context in which it is used.

Spectral imaging is a systems level challenge, in contrast to a
problem that can be solved with a detector strategy alone. A salient
point to consider is the strategy with which multidimensional
spectral data is acquired. As has already been observed, point
scanning instruments offer exquisite optical sectioning capability
with the disadvantage of fewer photons contributing to the overall
signal/pixel for a given rate of image acquisition, with a sample of
given quantum yield, at a given resolution and a given illumination
dosage. For truly unambiguous determination of the 3-D origin of a
spectral 'super-pixel', it could be argued that non-linear
(multiphoton) excitation provides significant advantages. Available
CCD based instruments collect information from many pixels in
parallel, so the integration time for each pixel at a given exposure
is longer and this provides the opportunity to collect more photons
at a given exposure time. Because CCD's are two-dimensional arrays of
detectors, many pixels of spatial information can be collected in
parallel along one dimension, while the entire spectra at each of
these pixels is dispersed across (and collected) in the second
dimension of the CCD chip...this is the paradigm that gives rise to
slit based, widefield illumination spectral detection systems. Such
instruments may collect an entire single row of pixels of spatial
information per exposure; in order to collect information from the
entire field of view, either the sample or the slit aperture is moved
to an adjacent position and another exposure is acquired. This
process is repeated until the entire dataset of 2 spatial dimensions
with spectral information at each pixel is acquired. But because the
widefield system is acquiring spatial data one row of pixels at a
time as opposed to one pixel at a time (as in the point scanning
instrument), the amount of time each pixel is permitted to integrate
photons for a given rate of acquisition is longer by a factor of how
ever many pixels there are in a row (assuming the same number of
pixels/row between instruments).

Yet another issue to consider is photobleaching and the possibility
of phototoxicity (where living samples are concerned). This is a
complex subject as well but it can be generalized that point scanning
instruments only illuminate single pixels as they are being imaged
(albiet at a high intensity) while a widefield instrument may
illuminate the entire field of view during the entire time the data-
set is being acquired. In sensitive samples, this may mean that the
entire field is photo-bleaching while pixels are only being imaged
one row at a time. One solution to this approach is to provide a
matching illumination slit in a plane confocal to the detection
aperture on a slit-based, widefield illumination spectral imaging
system. This restricts photobleaching to the row of pixels being
acquired and provides some rejection of extraneous light from out-of-
focus planes; there is at least one widefield system on the market
that takes this approach and couples it to a back-thinned EMCCD
detector.

There are several other approaches to the challenge of spectral
imaging as well: LCD filters that change color and permit collection
of 2-D images at different wavelengths in serial, point-scanning
instruments that collect spectral data in serial by changing the
detection range after each image in turn, frequency space fourier-
transform based spectral imaging devices, and, in the basic research
arena, a technology that uses a holographic grating to disperse
entire 2-D images across a large CCD to permit parallel collection of
spectral and 2-D spatial data. Presently the point scanning confocal
and line-scanning widefield approaches tend to be the most relevant
to fluorescence imaging applications in the biological sciences, the
best tool for the job depends on the relative importance of a number
of features/capabilities and each approach has it's merits.

Sincere Regards,
Karl

Karl Garsha
Head Application Scientist
Microimaging Applications Group (MAG)
3440 East Britannia Drive
Tucson, AZ 85629
tel 520.889.9933
direct 520.547.2704
fax 520.573.1944

MAG Biosystems| Photometrics| QImaging|
Media Cybernetics | Gatan
www.MAGbiosystems.com www.photomet.com
www.qimaging.comwww.mediacy.comwww.gatan
.com

The Microimaging Applications Group comprises five imaging leaders:
Photometrics, Gatan, Media Cybernetics,
QImaging, and MAG Biosystems. These technology partners work
independently as well as in synergy to offer an
unparalleled range of solutions for microimaging applications.

This message and any attachments are solely for the use of intended
recipients. They may contain privileged and/or confidential
information.
If you are not the intended recipient, you are hereby notified that
you received this email in error, and that any review, dissemination,
distribution
or copying of this email and any attachment is strictly prohibited.
If you receive this email in error please contact the sender and
delete the
message and any attachments associated therewith from your computer.
Your cooperation in this matter is appreciated.

Craig Brideau wrote:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

So what's the catch? Why do people still use PMTs vs. CCDs or
EMCCDs? Cost?

Craig

On 10/5/07, Kurt Thorn <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In general CCDs are more sensitive that PMTs. I can't speak to the
sensitivities of the systems here, but a typical PMT has a quantum
efficiency on the order of 10 - 25%, whereas good CCDs have QEs from
60
- 90%.

Kurt

Craig Brideau wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Will this system have the same sort of sensitivity as a multi-anode
PMT system like the C1si? This PARISS system seems to use a CCD
camera as its detector, which will not be as sensitive, if I
understand correctly?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Criag
If spectral analysis is the endpoint you may want to consider the
PARISS
system from Lightform. ( http://www.lightforminc.com/) that can be
placed on a widefield microscope.
We are using the PARISS system to obtain spectral data --it has 1nm
resolution and has a spectrum from 400-900nm. I find it to be a great
asset in the laboratory,
It is far more sensitive and accurate that any confocal system that I
have seen.

Contact Jeremy Lerner for details on his system.
best wishes
Bob

LightForm, Inc.,
601 Route 206, Suite 26-479
Hillsborough NJ 08844
Tel: (908)281 9098
Email: [hidden email]

.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

Craig Brideau
<craig.brideau@G
MAIL.COM>
To
Sent by: [hidden email]
Confocal
cc
Microscopy List
<CONFOCAL@LISTSE
Subject
RV.BUFFALO.EDU> Re: Nikon C1si?

10/05/2007 05:48
PM

Please respond
to
Confocal
Microscopy List
<CONFOCAL@LISTSE
RV.BUFFALO.EDU>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for your input! As in everything, there's a trade-off. Still,
for us the spectral data will be worth it.

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
The efficiency of detection is much less in the spectral mode due to

the

design of the multianode detector. In addition as Kurt Thorn said the
light is split into a number of different bandpass channels instead of
being detected by only one channel which also limits the detection in
each channel.
Nikon compensates for this decreased light by increasing the pixel

dwell

time. However photons are photons. With less photons you will get a
noisier image.
The spectral detection of any confocal system using a multianode
detector will not be as good as a PMT designed for good sensitivity

and

low light detection. It will produce nosier images but if there is
enough light you will be able to determine a valuable spectrum which

can

be used for scientific experiments and to manipulate images. Like
everything with confocal microscopy there is trade-offs and no perfect
system.
Best wishes.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

Craig Brideau
<craig.brideau@G
MAIL.COM>

To

Sent by: [hidden email]
Confocal

cc

Microscopy List
<CONFOCAL@LISTSE

Subject

RV.BUFFALO.EDU> Re: Nikon C1si?

10/04/2007 02:49
PM

Please respond
to
Confocal
Microscopy List
<CONFOCAL@LISTSE
RV.BUFFALO.EDU>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I was wondering if you saw this decreased throughput in spectral mode
only, or also in conventional detection mode? The spectral mode works
with a multi-anode PMT, which has a lower quantum efficiency than a
conventional PMT. From what I have seen of the design, it also has
the option to use a conventional PMT system with the device, and I am
wondering if you have also tried that mode of operation?

Thanks,

Craig

On 10/4/07, Robert Zucker <[hidden email]> wrote:

One of the major limitations that we have seen with this unit and

also

with the Zeiss meta 510 is the decreased light throughput . This
creates images that are noisier than conventional confocal

microscopes.

In our hands it appears you will need to have a bright sample to

make

the spectral system work properly. ,

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

UCSF MC 2140
Genentech Hall Room S252
600 16th St.
San Francisco, CA 94158-2517

http://nic.ucsf.edu
phone 415.514.9709
fax 415.514.4300

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

vb-2

Re: Nikon C1si?

In reply to this post by Karl Garsha

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks Karl for the posting and Jim for the book.

I agree that "sensitivity is a complex term". Could we make it rather simpler?

I was suggesting earlier to set up a company (Federally or "privately" funded),
or Genossenschaft (Swiss and German understand what I am talking about, and
both Swiss and German government is the Federal one, too) to answer millions of
questions remained illusive (The DOD should be another highly interested
party!!!), and everyone knows that Carl Zeiss is very hesitant to reveal
their "proprietary" specs; once they refused to reveal the QE of the AxioCam
CCD box:

Measure/compare:

All high end light detectors in rather detail, carefully evaluating/measuring
(with error bars) all sources of noise/artefacts in relation to both photon
detectors and microscope systems:
read-out noise, amplification noise a PMT, an EM-CCD, an EB-CCD; shot noise of
a number of scientific grade light detectors and so so so so on onnnnnnn....-
Karl and Jim have described it in great detail.

For example, among those millions mentioned above, there is another pending
task to do: to compare confocal, wide-field and TIR under conditions of low
light imaging (which confocal system lacks of an open pinhole artefact and
suitable for the (glass) surface imaging????)

Happy Columbus Day! I do hope we will discover something grand, beyond America!

Vitaly
NCI-Frederick,
301-846-6575

Quoting Karl Garsha <[hidden email]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>
>
>
>
>
> Hello all,
>
>
>
> There are several points to consider on the issue of sensitivity and
> the tradeoffs between signal to noise ratio and
> optical sectioning capability with regard to different genres of
> spectral imaging instrument, different approaches have different
> strengths and weaknesses.
>
>
>
> Sensitivity is a complex term. I think it is important to discern
> between quantum efficiency and amplification; detectors with high
> quantum efficiency get a high percentage of the available signal from a
> given optics train. Detectors with high amplification take the portion
> of available signal that is detected and amplify it to a much higher
> signature. As a general rule, scientific grade CCD detectors have
> higher quantum efficiency than PMT's. A typical front-illuminated,
> microlensed CCD will have a QE on the order of 60-70%, a back-thinned
> CCD may have a QE on the order of 90-95%, and a PMT's generally have a
> lower effective QE as Jim pointed out. However, conventional CCD's
> don't amplify the signal prior to readout, PMT's do (by as much as
> factor of a million times). EMCCD cameras are generally backthinned for
> high quantum efficiency and also amplify the signal prior to readout
> (this provides a benefit to the signal to noise ratio because the
> readout noise is not amplified with the signal), although the signal is
> amplified to a lesser extent than with PMT's. It is important to
> consider the sources of noise in addition to QE when discussing
> detector technologies, the signal-to-noise ratio is generally the
> concept people are trying to allude to when discussing sensitivity.
> Noise can arise from the physical nature of photon counting statistics
> (shot noise), the readout electronics (read noise), the signal
> amplification process (excess noise factor, spurious charge) and dark
> current. Jim Pawley's 'Handbook of Biological Confocal Microscopy, 3rd
> edition' discusses such concepts in depth and is an excellent resource.
> The
> proportional impact of different noise sources and the signal-to-noise
> ratio that can be achieved by a given detector technology depends on
> the technology itself convolved with the context in which it is used.
>
>
>
>
> Spectral imaging is a systems level challenge, in contrast to a problem
> that can be solved with a detector strategy alone. A salient point to
> consider is the strategy with which
> multidimensional spectral data is acquired. As has already been
> observed, point scanning instruments offer exquisite optical sectioning
> capability with the disadvantage of fewer photons contributing to the
> overall signal/pixel for a given rate of image acquisition, with a
> sample of given quantum yield, at a given resolution and a given
> illumination dosage. For truly unambiguous determination of the 3-D
> origin of a spectral 'super-pixel', it could be argued that non-linear
> (multiphoton) excitation provides significant advantages. Available CCD
> based instruments collect information from many pixels in parallel, so
> the integration time for each pixel at a given exposure is longer and
> this provides the opportunity to collect more photons at a given
> exposure time. Because CCD's are two-dimensional arrays of detectors,
> many pixels of spatial information can be collected in parallel along
> one dimension, while the entire spectra at each of these pixels is
> dispersed across (and collected) in the second dimension of the CCD
> chip...this is the paradigm that gives rise to slit based, widefield
> illumination spectral detection systems. Such instruments may collect
> an entire single row of pixels of spatial information per exposure; in
> order to collect information from the entire field of view, either the
> sample or the slit aperture is moved to an adjacent position and
> another exposure is acquired. This process is repeated until the entire
> dataset of 2 spatial dimensions with spectral information at each pixel
> is acquired. But because the widefield system is acquiring spatial data
> one row of pixels at a time as opposed to one pixel at a time (as in
> the point scanning instrument), the amount of time each pixel is
> permitted to integrate photons for a given rate of acquisition is
> longer by a factor of how ever many pixels there are in a row (assuming
> the same number of pixels/row between instruments).
>
>
>
> Yet another issue to consider is photobleaching and the possibility of
> phototoxicity (where living samples are concerned). This is a complex
> subject as well but it can be generalized that point scanning
> instruments only illuminate single pixels as they are being imaged
> (albiet at a high intensity) while a widefield instrument may
> illuminate the entire field of view during the entire time the data-set
> is being acquired. In sensitive samples, this may mean that the entire
> field is photo-bleaching while pixels are only being imaged one row at
> a time. One solution to this approach is to provide a matching
> illumination slit in a plane confocal to the detection aperture on a
> slit-based, widefield illumination spectral imaging system. This
> restricts photobleaching to the row of pixels being acquired and
> provides some rejection of extraneous light from out-of-focus planes;
> there is at least one widefield system on the market that takes this
> approach and couples it to a back-thinned EMCCD detector.
>
>
>
> There are several other approaches to the challenge of spectral imaging
> as well: LCD filters that change color and permit collection of 2-D
> images at different wavelengths in serial, point-scanning instruments
> that collect spectral data in serial by changing the detection range
> after each image in turn, frequency space fourier-transform based
> spectral imaging devices, and, in the basic research arena, a
> technology that uses a holographic grating to disperse entire 2-D
> images across a large CCD to permit parallel collection of spectral and
> 2-D spatial data. Presently the point scanning confocal and
> line-scanning widefield approaches tend to be the most relevant to
> fluorescence imaging applications in the biological sciences, the best
> tool for the job depends on the relative importance of a number of
> features/capabilities and each approach has it's merits.
>
>
>
> Sincere Regards,
>
> Karl
>
>
>
>
>
>
>
>
>
> MAG Signature
>
>
>
>
>
>
>
> Karl
> Garsha
>
> Head Application Scientist
>
> Microimaging Applications Group (MAG)
>
> 3440 East
> Britannia Drive
>
> Tucson,
> AZ
> 85629
>
> tel 520.889.9933
>
> direct 520.547.2704
>
> fax 520.573.1944
>
>
>
>
> MAG
> Biosystems
>     |     Photometrics
>     |     QImaging
>     |     Media Cybernetics     |
>     Gatan
>
>
> www.MAGbiosystems.com      www.photomet.com      www.qimaging.com
>            www.mediacy.com
>               www.gatan.com
>
>
> The
> Microimaging Applications Group comprises
> five
> imaging leaders: Photometrics, Gatan,
> Media Cybernetics,
>
> QImaging, and MAG
> Biosystems.
> These technology partners work independently as well as in
> synergy to offer an
>
> unparalleled range of solutions for
> microimaging
> applications.
>
>
>
>
>
> This
> message and any attachments are solely for the use of intended
> recipients. They
> may contain privileged and/or confidential information.
>
> If you are not the intended recipient, you are hereby notified that you
> received this email in error, and that any review, dissemination,
> distribution
>
> or copying of this email and any attachment is strictly prohibited. If
> you
> receive this email in error please contact the sender and delete the
>
> message and any attachments associated therewith from your computer.
> Your
> cooperation in this matter is appreciated.

-------------------------------------------------

Judy Trogadis

Re: Nikon C1si? -spectral unmixing

In reply to this post by Craig Brideau

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent practical references on spectral unmixing?

Thanks.
Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

>>> Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:

Julio Vazquez

Re: Nikon C1si? -spectral unmixing

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Hi Judy,

For starters, you could have a look at these:

1: Adv Biochem Eng Biotechnol. 2005;95:245-65.

Spectral imaging and linear unmixing in light microscopy.

Zimmermann T.

1: FEBS Lett. 2003 Jul 3;546(1):87-92.

Spectral imaging and its applications in live cell microscopy.

Zimmermann T, Rietdorf J, Pepperkok R.

1: Genes Cells. 2002 Sep;7(9):881-7.

Spectral imaging fluorescence microscopy.

Haraguchi T, Shimi T, Koujin T, Hashiguchi N, Hiraoka Y.

For a paper on various unmixing algorithms:

www.ll.mit.edu/news/journal/ pdf/vol14_no1/14_1suvery.pdf

(you can find it by Googling: spectral unmixing Keshava)

ImageJ has some unmixing plugins, with documentation, that may be useful:

http://rsb.info.nih.gov/ij/plugins/docs/SpectralUnmixing.pdf

Otherwise, if you search PubMed for "spectral unmixing" or "linear unmixing", you will find many references among which you can choose the ones that are most relevant to your interests...

Julio.

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

On Oct 8, 2007, at 8:59 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent practical references on spectral unmixing?

Thanks.

Judy

Lenaldo Rocha

Re: Nikon C1si? -spectral unmixing

In reply to this post by Judy Trogadis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Judy

You can read this one by Timo Zimmermann:

Zimmermann T, Rietdorf J, Pepperkok R. Spectral imaging and its
applications in live cell microscopy. FEBS Lett. 2003 Jul 3;546(1):87-92

[]

Lenaldo

Julio Vazquez

Re: Nikon C1si? -spectral unmixing

In reply to this post by Judy Trogadis

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

and I guess I should add:

Dickinson et al, Biotechniques 31: 1272-1278 (2001): Multi-spectral unmixing and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy

and

Zucker R and Lerner J, Cytometry 62A:8-34 (2004): Calibration and validation of confocal spectral imaging systems

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

[hidden email]

http://www.fhcrc.org

On Oct 8, 2007, at 8:59 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent practical references on spectral unmixing?

Thanks.
Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Craig,

The Lightform PARISS has a detection slit in front of the spectral
dispersion element and the CCD. This makes it half-confocal. If a matching
slit is placed in the epi-illumination field aperture plane (or a conjugate
plane) it is a slit confocal (aka line scan confocal). The result is an
X*Spectral image. If the microscope has a motorized stage, the Y-axis can be
added = push broom confocal imaging spectrometer. The performance is such
that the emission filter can be removed from the epi-illumination filter
cube when used with an Hg lamp (the bright Hg line or lines are one
nanometer or so side = 1 or a few pixels, so do not bleed into the emission
areas and can be used to quantify the excitation power). The PARISS can also
be used for brightfield images.

You can contact Jeremy Lerner at http://www.lightforminc.com/ for
additional details about the PARISS.

With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point
scanning confocal microscope detects a maximum of about 8 photons per pixel
per scan. This is a nice match to a PMT (or APD for far red and infrared
photons), not so good for a CCD (see Jim's post about noise). On the other
hand, a CCD is an area detector (or line * spectrum detector for the
PARISS), so the CCD is a great way to acquire 2D images fast. A CCD or EMCCD
system can be used to image a thin optical section, quickly, with judicious
use of excitation light, as in TIRF, fast multipoint multiphoton scanners
such as the TriMScope (if it can be made to work reliably) or Scherer's
stochastic scanner (
http://www.opticsexpress.org/abstract.cfm?id=89328), or
hopefully by widefield multiphoton excitation (Brooker 2007 US patent
7,170,675 ), or by multiplane acquisition followed by digital deconvolution.
If you can buy or borrow 3 EMCCD's like Ian Parker, you can acquire multiple
focal planes simultaneously, see PubMed 17716727, also similar rig by
Prabhat, PubMed 17384151.

Back to 32-channel PMT confocal microscopes: the Zeiss META detector
includes blockers for the major laser lines. As far as I am aware, they are
in the light path all the time (the 488 nm blocker may be responsible for
zigzag artifacts in CFP emission spectra, though bad PMT channels could also
be responsible). Nikon may include similar blockers in the si. The Zeiss
LSM510 light path can be configured to use a microscope filter cube. With
the right cube and a mirror slide, you can acquire the spectrum of the Hg
lamp using the META, or you could buy a Lightform MIDL like Bob Zucker's and
just lay it on the microscope. The META spectrum of the MIDL lamp is not
even close to the calibration curve that comes with the MIDL. Bob and Jeremy
have published the close match of the PARISS data for the MIDL.

Enjoy,

George

At 07:26 PM 10/5/2007, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks! We are interested in co-localization, for which we sometimes
need the third dimension. Mainly we want to do spectral unmixing for
multiple dyes and colocalization, so we really do need optical
slicing!

Thanks,

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
you are correct.
Currently the PARISS only detects X-Y--not depth.
however, most data that I have seen from confocal spectral systems are
only a single XY slice. they usually are not used to do a 3D
reconstruction in spectral mode.
bob
.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by: [hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

   Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:44
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It doesn't seem to deal with depth though? It only detects in X-Y, so
can it generate optical slices?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The PARISS system uses a Retiga 2000R cooled CCD camera. It is far

more

sensitive and accurate than any confocal microscope.
You can detect things in that system that can not be observed with a
confocal spectral system. It is a great system in my opinion.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by: [hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:19
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Will this system have the same sort of sensitivity as a multi-anode
PMT system like the C1si? This PARISS system seems to use a CCD
camera as its detector, which will not be as sensitive, if I
understand correctly?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Criag
If spectral analysis is the endpoint you may want to consider the

PARISS

system from Lightform. ( http://www.lightforminc.com/) that can be
placed on a widefield microscope.
We are using the PARISS system to obtain spectral data --it has 1nm
resolution and has a spectrum from 400-900nm. I find it to be a

great

asset in the laboratory,
It is far more sensitive and accurate that any confocal system that

I

have seen.

Contact Jeremy Lerner for details on his system.
best wishes
Bob

LightForm, Inc.,
601 Route 206, Suite 26-479
   Hillsborough NJ 08844
   Tel: (908)281 9098
Email: [hidden email]

.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by: [hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 05:48
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for your input! As in everything, there's a trade-off.

Still,

for us the spectral data will be worth it.

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
The efficiency of detection is much less in the spectral mode due

to

the

design of the multianode detector. In addition as Kurt Thorn said

the

light is split into a number of different bandpass channels

instead

of

being detected by only one channel which also limits the detection

in

each channel.
Nikon compensates for this decreased light by increasing the pixel

dwell

time. However photons are photons. With less photons you will get

a

noisier image.
The spectral detection of any confocal system using a multianode
detector will not be as good as a PMT designed for good

sensitivity

and

low light detection. It will produce nosier images but if there is
enough light you will be able to determine a valuable spectrum

which

can

be used for scientific experiments and to manipulate images. Like
everything with confocal microscopy there is trade-offs and no

perfect

system.
Best wishes.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by:

[hidden email]

   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/04/2007 02:49
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I was wondering if you saw this decreased throughput in spectral

mode

only, or also in conventional detection mode? The spectral mode

works

with a multi-anode PMT, which has a lower quantum efficiency than

a

conventional PMT. From what I have seen of the design, it also

has

the option to use a conventional PMT system with the device, and I

am

wondering if you have also tried that mode of operation?

Thanks,

Craig

On 10/4/07, Robert Zucker <[hidden email]> wrote:

One of the major limitations that we have seen with this unit

and

also

with the Zeiss meta 510 is the decreased light throughput .

This

creates images that are noisier than conventional confocal

microscopes.

In our hands it appears you will need to have a bright sample to

make

the spectral system work properly. ,

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp
(see Analytical Imaging Core Facility)

Judy Trogadis

Re: Nikon C1si? -spectral unmixing

In reply to this post by Judy Trogadis

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for the reference - I received another one - but I am looking for a real live application paper, not a review of the technique. Years ago I attended a lecture by Ken Spring on FRET - he said "everybody wants to do FRET, even the janitor wants to do FRET". He claimed there were more review papers on the technique than actual original data papers. So, actually, I'm looking for application papers with original data.

Thanks, hope I have not said anything inappropriate towards all the technique papers - they are an essential background.

Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

>>> Julio Vazquez <[hidden email]> 10/08/07 1:28 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

=

and I guess I should add:

Dickinson et al, Biotechniques 31: 1272-1278 (2001): Multi-spectral
unmixing and linear unmixing add a whole new dimension to laser
scanning fluorescence microscopy

and

Zucker R and Lerner J, Cytometry 62A:8-34 (2004): Calibration and
validation of confocal spectral imaging systems

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

[hidden email]
http://www.fhcrc.org

On Oct 8, 2007, at 8:59 AM, Judy Trogadis wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> In light of this discussion, can anyone suggest some recent
> practical references on spectral unmixing?
>
> Thanks.
> Judy
>
>
>
> Judy Trogadis
> Bio-Imaging Coordinator
> St. Michael's Hospital, 7Queen
> 30 Bond St.
> Toronto, ON M5B 1W8, Canada
> ph: 416-864-6060 x6337
> pager: 416-685-9219
> fax: 416-864-6043
> [hidden email]
>
>
>>>> Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> You make some interesting points! It does seem that the low threshold
> capability of PMT's make them more useful for our photon counting type
> experiments. Still, in the case of a brighter sample the speed of an
> EMCCD would be useful.
>
> Thanks,
>
> Craig
>
> On 10/6/07, George McNamara <[hidden email]> wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> Hi Craig,
>>
>> The Lightform PARISS has a detection slit in front of the spectral
>> dispersion element and the CCD. This makes it half-confocal. If a
>> matching
>> slit is placed in the epi-illumination field aperture plane (or a
>> conjugate
>> plane) it is a slit confocal (aka line scan confocal). The result
>> is an
>> X*Spectral image. If the microscope has a motorized stage, the Y-
>> axis can be
>> added = push broom confocal imaging spectrometer. The performance
>> is such
>> that the emission filter can be removed from the epi-illumination
>> filter
>> cube when used with an Hg lamp (the bright Hg line or lines are one
>> nanometer or so side = 1 or a few pixels, so do not bleed into the
>> emission
>> areas and can be used to quantify the excitation power). The
>> PARISS can also
>> be used for brightfield images.
>>
>> You can contact Jeremy Lerner at http://www.lightforminc.com/ for
>> additional details about the PARISS.
>>
>>
>> With respect to PMT vs CCD or EMCCD, Pawley's rule is that a
>> typical point
>> scanning confocal microscope detects a maximum of about 8 photons
>> per pixel
>> per scan. This is a nice match to a PMT (or APD for far red and
>> infrared
>> photons), not so good for a CCD (see Jim's post about noise). On
>> the other
>> hand, a CCD is an area detector (or line * spectrum detector for the
>> PARISS), so the CCD is a great way to acquire 2D images fast. A
>> CCD or EMCCD
>> system can be used to image a thin optical section, quickly, with
>> judicious
>> use of excitation light, as in TIRF, fast multipoint multiphoton
>> scanners
>> such as the TriMScope (if it can be made to work reliably) or
>> Scherer's
>> stochastic scanner (
>> http://www.opticsexpress.org/abstract.cfm?id=89328), or
>> hopefully by widefield multiphoton excitation (Brooker 2007 US patent
>> 7,170,675 ), or by multiplane acquisition followed by digital
>> deconvolution.
>> If you can buy or borrow 3 EMCCD's like Ian Parker, you can
>> acquire multiple
>> focal planes simultaneously, see PubMed 17716727, also similar rig by
>> Prabhat, PubMed 17384151.
>>
>> Back to 32-channel PMT confocal microscopes: the Zeiss META detector
>> includes blockers for the major laser lines. As far as I am aware,
>> they are
>> in the light path all the time (the 488 nm blocker may be
>> responsible for
>> zigzag artifacts in CFP emission spectra, though bad PMT channels
>> could also
>> be responsible). Nikon may include similar blockers in the si. The
>> Zeiss
>> LSM510 light path can be configured to use a microscope filter
>> cube. With
>> the right cube and a mirror slide, you can acquire the spectrum of
>> the Hg
>> lamp using the META, or you could buy a Lightform MIDL like Bob
>> Zucker's and
>> just lay it on the microscope. The META spectrum of the MIDL lamp
>> is not
>> even close to the calibration curve that comes with the MIDL. Bob
>> and Jeremy
>> have published the close match of the PARISS data for the MIDL.
>>
>>
>> Enjoy,
>>
>>
>> George
>>
>>
>>
>> At 07:26 PM 10/5/2007, you wrote:
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Thanks! We are interested in co-localization, for which we
>> sometimes
>> need the third dimension. Mainly we want to do spectral unmixing
>> for
>> multiple dyes and colocalization, so we really do need optical
>> slicing!
>>
>> Thanks,
>>
>> Craig
>>
>>
>> On 10/5/07, Robert Zucker <[hidden email]> wrote:
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> Craig
>>> you are correct.
>>> Currently the PARISS only detects X-Y--not depth.
>>> however, most data that I have seen from confocal spectral
>>> systems are
>>> only a single XY slice. they usually are not used to do a 3D
>>> reconstruction in spectral mode.
>>> bob
>>> .
>>>
>>>
>>> Robert M. Zucker, PhD
>>> U.S. Environmental Protection Agency
>>> Office of Research and Development
>>> National Health and Environmental Effects Research Laboratory.
>>> Telephone: 919-541-1585 Fax: 919-541-4017
>>> e-mail: [hidden email]
>>>
>>> Mail address: Reproductive Toxicology Division, MD 67
>>> 2525 E.NC Highway 54
>>> Research Triangle Park, North Carolina, 27711
>>>
>>> Shipping address: 2525 E.NC Highway 54
>>> Durham, NC, 27713
>>>
>>>
>>>
>>>
>>> Craig Brideau
>>> <craig.brideau@G
>>> MAIL.COM>
>> To
>>> Sent by: [hidden email]
>>> Confocal
>> cc
>>> Microscopy List
>>> <CONFOCAL@LISTSE
>> Subject
>>> RV.BUFFALO.EDU> Re: Nikon C1si?
>>>
>>>
>>> 10/05/2007 06:44
>>> PM
>>>
>>>
>>> Please respond
>>> to
>>> Confocal
>>> Microscopy List
>>> <CONFOCAL@LISTSE
>>> RV.BUFFALO.EDU>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Search the CONFOCAL archive at
>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>
>>> It doesn't seem to deal with depth though? It only detects in X-
>>> Y, so
>>> can it generate optical slices?
>>>
>>> Craig
>>>
>>> On 10/5/07, Robert Zucker <[hidden email]> wrote:
>>>> Search the CONFOCAL archive at
>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>> The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
>>> more
>>>> sensitive and accurate than any confocal microscope.
>>>> You can detect things in that system that can not be observed
>>>> with a
>>>> confocal spectral system. It is a great system in my opinion.
>>>> Bob
>>>>
>>>> Robert M. Zucker, PhD
>>>> U.S. Environmental Protection Agency
>>>> Office of Research and Development
>>>> National Health and Environmental Effects Research Laboratory.
>>>> Telephone: 919-541-1585 Fax: 919-541-4017
>>>> e-mail: [hidden email]
>>>>
>>>> Mail address: Reproductive Toxicology Division, MD 67
>>>> 2525 E.NC Highway 54
>>>> Research Triangle Park, North Carolina, 27711
>>>>
>>>> Shipping address: 2525 E.NC Highway 54
>>>> Durham, NC, 27713
>>>>
>>>>
>>>>
>>>>
>>>> Craig Brideau
>>>> <craig.brideau@G
>>>> MAIL.COM>
>>> To
>>>> Sent by: [hidden email]
>>>> Confocal
>>> cc
>>>> Microscopy List
>>>> <CONFOCAL@LISTSE
>>> Subject
>>>> RV.BUFFALO.EDU> Re: Nikon C1si?
>>>>
>>>>
>>>> 10/05/2007 06:19
>>>> PM
>>>>
>>>>
>>>> Please respond
>>>> to
>>>> Confocal
>>>> Microscopy List
>>>> <CONFOCAL@LISTSE
>>>> RV.BUFFALO.EDU>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> Search the CONFOCAL archive at
>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>
>>>> Will this system have the same sort of sensitivity as a multi-anode
>>>> PMT system like the C1si? This PARISS system seems to use a CCD
>>>> camera as its detector, which will not be as sensitive, if I
>>>> understand correctly?
>>>>
>>>> Craig
>>>>
>>>> On 10/5/07, Robert Zucker <[hidden email]> wrote:
>>>>> Search the CONFOCAL archive at
>>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>> Criag
>>>>> If spectral analysis is the endpoint you may want to consider the
>>>> PARISS
>>>>> system from Lightform. ( http://www.lightforminc.com/) that can be
>>>>> placed on a widefield microscope.
>>>>> We are using the PARISS system to obtain spectral data --it has
>>>>> 1nm
>>>>> resolution and has a spectrum from 400-900nm. I find it to be a
>>> great
>>>>> asset in the laboratory,
>>>>> It is far more sensitive and accurate that any confocal system
>>>>> that
>>> I
>>>>> have seen.
>>>>>
>>>>> Contact Jeremy Lerner for details on his system.
>>>>> best wishes
>>>>> Bob
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> LightForm, Inc.,
>>>>> 601 Route 206, Suite 26-479
>>>>> Hillsborough NJ 08844
>>>>> Tel: (908)281 9098
>>>>> Email: [hidden email]
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> .
>>>>>
>>>>> Robert M. Zucker, PhD
>>>>> U.S. Environmental Protection Agency
>>>>> Office of Research and Development
>>>>> National Health and Environmental Effects Research Laboratory.
>>>>> Telephone: 919-541-1585 Fax: 919-541-4017
>>>>> e-mail: [hidden email]
>>>>>
>>>>> Mail address: Reproductive Toxicology Division, MD 67
>>>>> 2525 E.NC Highway 54
>>>>> Research Triangle Park, North Carolina, 27711
>>>>>
>>>>> Shipping address: 2525 E.NC Highway 54
>>>>> Durham, NC, 27713
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> Craig Brideau
>>>>> <craig.brideau@G
>>>>> MAIL.COM>
>>>> To
>>>>> Sent by:
>>>>> [hidden email]
>>>>> Confocal
>>>> cc
>>>>> Microscopy List
>>>>> <CONFOCAL@LISTSE
>>>> Subject
>>>>> RV.BUFFALO.EDU> Re: Nikon C1si?
>>>>>
>>>>>
>>>>> 10/05/2007 05:48
>>>>> PM
>>>>>
>>>>>
>>>>> Please respond
>>>>> to
>>>>> Confocal
>>>>> Microscopy List
>>>>> <CONFOCAL@LISTSE
>>>>> RV.BUFFALO.EDU>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> Search the CONFOCAL archive at
>>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>
>>>>> Thanks for your input! As in everything, there's a trade-off.
>>> Still,
>>>>> for us the spectral data will be worth it.
>>>>>
>>>>> Craig
>>>>>
>>>>> On 10/5/07, Robert Zucker <[hidden email]> wrote:
>>>>>> Search the CONFOCAL archive at
>>>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>>
>>>>>> Craig
>>>>>> The efficiency of detection is much less in the spectral mode due
>>> to
>>>>> the
>>>>>> design of the multianode detector. In addition as Kurt Thorn said
>>>> the
>>>>>> light is split into a number of different bandpass channels
>>> instead
>>>> of
>>>>>> being detected by only one channel which also limits the
>>>>>> detection
>>>> in
>>>>>> each channel.
>>>>>> Nikon compensates for this decreased light by increasing the
>>>>>> pixel
>>>>> dwell
>>>>>> time. However photons are photons. With less photons you will get
>>> a
>>>>>> noisier image.
>>>>>> The spectral detection of any confocal system using a multianode
>>>>>> detector will not be as good as a PMT designed for good
>>> sensitivity
>>>>> and
>>>>>> low light detection. It will produce nosier images but if
>>>>>> there is
>>>>>> enough light you will be able to determine a valuable spectrum
>>> which
>>>>> can
>>>>>> be used for scientific experiments and to manipulate images. Like
>>>>>> everything with confocal microscopy there is trade-offs and no
>>>> perfect
>>>>>> system.
>>>>>> Best wishes.
>>>>>> Bob
>>>>>>
>>>>>> Robert M. Zucker, PhD
>>>>>> U.S. Environmental Protection Agency
>>>>>> Office of Research and Development
>>>>>> National Health and Environmental Effects Research Laboratory.
>>>>>> Telephone: 919-541-1585 Fax: 919-541-4017
>>>>>> e-mail: [hidden email]
>>>>>>
>>>>>> Mail address: Reproductive Toxicology Division, MD 67
>>>>>> 2525 E.NC Highway 54
>>>>>> Research Triangle Park, North Carolina, 27711
>>>>>>
>>>>>> Shipping address: 2525 E.NC Highway 54
>>>>>> Durham, NC, 27713
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Craig Brideau
>>>>>> <craig.brideau@G
>>>>>> MAIL.COM>
>>>>> To
>>>>>> Sent by:
>>> [hidden email]
>>>>>> Confocal
>>>>> cc
>>>>>> Microscopy List
>>>>>> <CONFOCAL@LISTSE
>>>>> Subject
>>>>>> RV.BUFFALO.EDU> Re: Nikon C1si?
>>>>>>
>>>>>>
>>>>>> 10/04/2007 02:49
>>>>>> PM
>>>>>>
>>>>>>
>>>>>> Please respond
>>>>>> to
>>>>>> Confocal
>>>>>> Microscopy List
>>>>>> <CONFOCAL@LISTSE
>>>>>> RV.BUFFALO.EDU>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> Search the CONFOCAL archive at
>>>>>>
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>>>>>
>>>>>> I was wondering if you saw this decreased throughput in spectral
>>>> mode
>>>>>> only, or also in conventional detection mode? The spectral mode
>>>> works
>>>>>> with a multi-anode PMT, which has a lower quantum efficiency than
>>> a
>>>>>> conventional PMT. From what I have seen of the design, it also
>>> has
>>>>>> the option to use a conventional PMT system with the device,
>>>>>> and I
>>>> am
>>>>>> wondering if you have also tried that mode of operation?
>>>>>>
>>>>>> Thanks,
>>>>>>
>>>>>> Craig
>>>>>>
>>>>>> On 10/4/07, Robert Zucker <[hidden email]> wrote:
>>>>>>> One of the major limitations that we have seen with this unit
>>> and
>>>>> also
>>>>>>> with the Zeiss meta 510 is the decreased light throughput .
>>> This
>>>>>>> creates images that are noisier than conventional confocal
>>>>>> microscopes.
>>>>>>> In our hands it appears you will need to have a bright sample to
>>>>> make
>>>>>>> the spectral system work properly. ,
>>>>>>
>>>>>
>>>>
>>>
>>
>>
>>
>>
>>
>>
>> George McNamara, Ph.D.
>> University of Miami, Miller School of Medicine
>> Image Core
>> Miami, FL 33010
>> [hidden email]
>> [hidden email]
>> 305-243-8436 office
>> http://home.earthlink.net/~pubspectra/
>> http://home.earthlink.net/~geomcnamara/
>> http://www.sylvester.org/health_pro/shared_resources/index.asp
>> (see Analytical Imaging Core Facility)
>>
>>
>>

Julio Vazquez

Re: Nikon C1si? -spectral unmixing

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Hi Judy,

Perhaps some of these?:

Julio.

1: Lansford R, Bearman G, Fraser SE.

Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy.

J Biomed Opt. 2001 Jul;6(3):311-8.

1: Cytometry A. 2006 Aug 1;69(8):904-11.

Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation.

Thaler C, Vogel SS.

1: Biophys J. 2005 Oct;89(4):2736-49. Epub 2005 Jul 22.

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Thaler C, Koushik SV, Blank PS, Vogel SS.

1: J Microsc. 2004 Aug;215(Pt 2):162-73.

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.

Gu Y, Di WL, Kelsell DP, Zicha D.

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

On Oct 8, 2007, at 11:37 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for the reference - I received another one - but I am looking for a real live application paper, not a review of the technique. Years ago I attended a lecture by Ken Spring on FRET - he said "everybody wants to do FRET, even the janitor wants to do FRET". He claimed there were more review papers on the technique than actual original data papers. So, actually, I'm looking for application papers with original data.

Thanks, hope I have not said anything inappropriate towards all the technique papers - they are an essential background.

Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

Julio Vazquez <[hidden email]> 10/08/07 1:28 PM >>>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

=

and I guess I should add:

Dickinson et al, Biotechniques 31: 1272-1278 (2001): Multi-spectral
unmixing and linear unmixing add a whole new dimension to laser
scanning fluorescence microscopy

and

Zucker R and Lerner J, Cytometry 62A:8-34 (2004): Calibration and
validation of confocal spectral imaging systems

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024

[hidden email]
http://www.fhcrc.org

On Oct 8, 2007, at 8:59 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent
practical references on spectral unmixing?

Thanks.
Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Craig,

The Lightform PARISS has a detection slit in front of the spectral
dispersion element and the CCD. This makes it half-confocal. If a
matching
slit is placed in the epi-illumination field aperture plane (or a
conjugate
plane) it is a slit confocal (aka line scan confocal). The result
is an
X*Spectral image. If the microscope has a motorized stage, the Y-
axis can be
added = push broom confocal imaging spectrometer. The performance
is such
that the emission filter can be removed from the epi-illumination
filter
cube when used with an Hg lamp (the bright Hg line or lines are one
nanometer or so side = 1 or a few pixels, so do not bleed into the
emission
areas and can be used to quantify the excitation power). The
PARISS can also
be used for brightfield images.

You can contact Jeremy Lerner at http://www.lightforminc.com/ for
additional details about the PARISS.

With respect to PMT vs CCD or EMCCD, Pawley's rule is that a
typical point
scanning confocal microscope detects a maximum of about 8 photons
per pixel
per scan. This is a nice match to a PMT (or APD for far red and
infrared
photons), not so good for a CCD (see Jim's post about noise). On
the other
hand, a CCD is an area detector (or line * spectrum detector for the
PARISS), so the CCD is a great way to acquire 2D images fast. A
CCD or EMCCD
system can be used to image a thin optical section, quickly, with
judicious
use of excitation light, as in TIRF, fast multipoint multiphoton
scanners
such as the TriMScope (if it can be made to work reliably) or
Scherer's
stochastic scanner (
http://www.opticsexpress.org/abstract.cfm?id=89328), or
hopefully by widefield multiphoton excitation (Brooker 2007 US patent
7,170,675 ), or by multiplane acquisition followed by digital
deconvolution.
If you can buy or borrow 3 EMCCD's like Ian Parker, you can
acquire multiple
focal planes simultaneously, see PubMed 17716727, also similar rig by
Prabhat, PubMed 17384151.

Back to 32-channel PMT confocal microscopes: the Zeiss META detector
includes blockers for the major laser lines. As far as I am aware,
they are
in the light path all the time (the 488 nm blocker may be
responsible for
zigzag artifacts in CFP emission spectra, though bad PMT channels
could also
be responsible). Nikon may include similar blockers in the si. The
Zeiss
LSM510 light path can be configured to use a microscope filter
cube. With
the right cube and a mirror slide, you can acquire the spectrum of
the Hg
lamp using the META, or you could buy a Lightform MIDL like Bob
Zucker's and
just lay it on the microscope. The META spectrum of the MIDL lamp
is not
even close to the calibration curve that comes with the MIDL. Bob
and Jeremy
have published the close match of the PARISS data for the MIDL.

Enjoy,

George

At 07:26 PM 10/5/2007, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks! We are interested in co-localization, for which we
sometimes
need the third dimension. Mainly we want to do spectral unmixing
for
multiple dyes and colocalization, so we really do need optical
slicing!

Thanks,

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
you are correct.
Currently the PARISS only detects X-Y--not depth.
however, most data that I have seen from confocal spectral
systems are
only a single XY slice. they usually are not used to do a 3D
reconstruction in spectral mode.
bob
.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by: [hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

   Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:44
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It doesn't seem to deal with depth though? It only detects in X-
Y, so
can it generate optical slices?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The PARISS system uses a Retiga 2000R cooled CCD camera. It is far

more

sensitive and accurate than any confocal microscope.
You can detect things in that system that can not be observed
with a
confocal spectral system. It is a great system in my opinion.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by: [hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:19
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Will this system have the same sort of sensitivity as a multi-anode
PMT system like the C1si? This PARISS system seems to use a CCD
camera as its detector, which will not be as sensitive, if I
understand correctly?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Criag
If spectral analysis is the endpoint you may want to consider the

PARISS

system from Lightform. ( http://www.lightforminc.com/) that can be
placed on a widefield microscope.
We are using the PARISS system to obtain spectral data --it has
1nm
resolution and has a spectrum from 400-900nm. I find it to be a

great

asset in the laboratory,
It is far more sensitive and accurate that any confocal system
that

I

have seen.

Contact Jeremy Lerner for details on his system.
best wishes
Bob

LightForm, Inc.,
601 Route 206, Suite 26-479
   Hillsborough NJ 08844
   Tel: (908)281 9098
Email: [hidden email]

.

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by:
[hidden email]
   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 05:48
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for your input! As in everything, there's a trade-off.

Still,

for us the spectral data will be worth it.

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig
The efficiency of detection is much less in the spectral mode due

to

the

design of the multianode detector. In addition as Kurt Thorn said

the

light is split into a number of different bandpass channels

instead

of

being detected by only one channel which also limits the
detection

in

each channel.
Nikon compensates for this decreased light by increasing the
pixel

dwell

time. However photons are photons. With less photons you will get

a

noisier image.
The spectral detection of any confocal system using a multianode
detector will not be as good as a PMT designed for good

sensitivity

and

low light detection. It will produce nosier images but if
there is
enough light you will be able to determine a valuable spectrum

which

can

be used for scientific experiments and to manipulate images. Like
everything with confocal microscopy there is trade-offs and no

perfect

system.
Best wishes.
Bob

Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory.
Telephone: 919-541-1585 Fax: 919-541-4017
e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67
2525 E.NC Highway 54
Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54
Durham, NC, 27713

   Craig Brideau
   <craig.brideau@G
   MAIL.COM>

To

   Sent by:

[hidden email]

   Confocal

cc

   Microscopy List
   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/04/2007 02:49
   PM

Please respond
to
   Confocal
   Microscopy List
   <CONFOCAL@LISTSE
   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I was wondering if you saw this decreased throughput in spectral

mode

only, or also in conventional detection mode? The spectral mode

works

with a multi-anode PMT, which has a lower quantum efficiency than

a

conventional PMT. From what I have seen of the design, it also

has

the option to use a conventional PMT system with the device,
and I

am

wondering if you have also tried that mode of operation?

Thanks,

Craig

On 10/4/07, Robert Zucker <[hidden email]> wrote:

One of the major limitations that we have seen with this unit

and

also

with the Zeiss meta 510 is the decreased light throughput .

This

creates images that are noisier than conventional confocal

microscopes.

In our hands it appears you will need to have a bright sample to

make

the spectral system work properly. ,

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/health_pro/shared_resources/index.asp
(see Analytical Imaging Core Facility)

Karl Garsha

Re: Nikon C1si? -spectral unmixing

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Here are several more application references addressing spectral detection in the context of FRET determination:

      Ecker, RC, de Martin, R, Steiner, GE, and Schmid, JA. (2004). Application of Spectral Imaging Microscopy in Cytomics and Fluorescence Resonance Energy Transfer (FRET) Analysis. Cytometry Part A 59A: 172-181.

      Raicu, V, Jansma, DB, Dwayne Miller, RJ, and Friesen, JD. (2005). Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer. Biochem. J. 385: 265-277.

      St. Croix, CM, Stitt, MS, Leelavanichkul, K, Wasserloos, KJ, Pitt, BR, and Watkins, SC. (2004). Nitric Oxide-Induced Modification of Protein Thiolate Clusters as Determined by Spectral Fluorescence Resonance Energy Transfer in Live Endothelial Cells. Free Radical Biology and Medicine 37, 6: 785-792.

      van Kuppeveld, FJM, Melchers, WJG, Willems, PHGM, and Gadella Jr., TWJ. (2002). Homomultimerization of the Coxsackievirus 2B Protein in Living Cells Visualized by Fluorescence Resonance Energy Transfer Microscopy. J. Virol. 76, 18: 9446-9456.

      Zimmermann, T, Rietdorf, J, Girod, A, Georget, V, and Pepperkok, R. (2002). Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531: 245-249.

Regards,
Karl

MAG Signature

Karl Garsha
Head Application Scientist
Microimaging Applications Group (MAG)
3440 East Britannia Drive
Tucson, AZ 85629
tel 520.889.9933
direct 520.547.2704
fax 520.573.1944

MAG Biosystems | Photometrics | QImaging | Media Cybernetics | Gatan
www.MAGbiosystems.com www.photomet.com www.qimaging.com www.mediacy.com www.gatan.com

Julio Vazquez wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Hi Judy,

Perhaps some of these?:

Julio.

==

1: Lansford R, Bearman G, Fraser SE.

Resolution of multiple green fluorescent protein color variants and dyes using two-photon microscopy and imaging spectroscopy.

J Biomed Opt. 2001 Jul;6(3):311-8.

==

1: Cytometry A. 2006 Aug 1;69(8):904-11.

Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation.

Thaler C, Vogel SS.

==

1: Biophys J. 2005 Oct;89(4):2736-49. Epub 2005 Jul 22.

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Thaler C, Koushik SV, Blank PS, Vogel SS.

==

1: J Microsc. 2004 Aug;215(Pt 2):162-73.

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.

Gu Y, Di WL, Kelsell DP, Zicha D.

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

On Oct 8, 2007, at 11:37 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for the reference - I received another one - but I am looking for a real live application paper, not a review of the technique. Years ago I attended a lecture by Ken Spring on FRET - he said "everybody wants to do FRET, even the janitor wants to do FRET". He claimed there were more review papers on the technique than actual original data papers. So, actually, I'm looking for application papers with original data.

Thanks, hope I have not said anything inappropriate towards all the technique papers - they are an essential background.

Judy

Judy Trogadis

Bio-Imaging Coordinator

St. Michael's Hospital, 7Queen

30 Bond St.

Toronto, ON M5B 1W8, Canada

ph: 416-864-6060 x6337

pager: 416-685-9219

fax: 416-864-6043

[hidden email]

Julio Vazquez <[hidden email]> 10/08/07 1:28 PM >>>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

=

and I guess I should add:

Dickinson et al, Biotechniques 31: 1272-1278 (2001): Multi-spectral

unmixing and linear unmixing add a whole new dimension to laser

scanning fluorescence microscopy

and

Zucker R and Lerner J, Cytometry 62A:8-34 (2004): Calibration and

validation of confocal spectral imaging systems

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

[hidden email]

http://www.fhcrc.org

On Oct 8, 2007, at 8:59 AM, Judy Trogadis wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent

practical references on spectral unmixing?

Thanks.

Judy

Judy Trogadis

Bio-Imaging Coordinator

St. Michael's Hospital, 7Queen

30 Bond St.

Toronto, ON M5B 1W8, Canada

ph: 416-864-6060 x6337

pager: 416-685-9219

fax: 416-864-6043

[hidden email]

Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold

capability of PMT's make them more useful for our photon counting type

experiments. Still, in the case of a brighter sample the speed of an

EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Craig,

The Lightform PARISS has a detection slit in front of the spectral

dispersion element and the CCD. This makes it half-confocal. If a

matching

slit is placed in the epi-illumination field aperture plane (or a

conjugate

plane) it is a slit confocal (aka line scan confocal). The result

is an

X*Spectral image. If the microscope has a motorized stage, the Y-

axis can be

added = push broom confocal imaging spectrometer. The performance

is such

that the emission filter can be removed from the epi-illumination

filter

cube when used with an Hg lamp (the bright Hg line or lines are one

nanometer or so side = 1 or a few pixels, so do not bleed into the

emission

areas and can be used to quantify the excitation power). The

PARISS can also

be used for brightfield images.

You can contact Jeremy Lerner at http://www.lightforminc.com/ for

additional details about the PARISS.

With respect to PMT vs CCD or EMCCD, Pawley's rule is that a

typical point

scanning confocal microscope detects a maximum of about 8 photons

per pixel

per scan. This is a nice match to a PMT (or APD for far red and

infrared

photons), not so good for a CCD (see Jim's post about noise). On

the other

hand, a CCD is an area detector (or line * spectrum detector for the

PARISS), so the CCD is a great way to acquire 2D images fast. A

CCD or EMCCD

system can be used to image a thin optical section, quickly, with

judicious

use of excitation light, as in TIRF, fast multipoint multiphoton

scanners

such as the TriMScope (if it can be made to work reliably) or

Scherer's

stochastic scanner (

http://www.opticsexpress.org/abstract.cfm?id=89328), or

hopefully by widefield multiphoton excitation (Brooker 2007 US patent

7,170,675 ), or by multiplane acquisition followed by digital

deconvolution.

If you can buy or borrow 3 EMCCD's like Ian Parker, you can

acquire multiple

focal planes simultaneously, see PubMed 17716727, also similar rig by

Prabhat, PubMed 17384151.

Back to 32-channel PMT confocal microscopes: the Zeiss META detector

includes blockers for the major laser lines. As far as I am aware,

they are

in the light path all the time (the 488 nm blocker may be

responsible for

zigzag artifacts in CFP emission spectra, though bad PMT channels

could also

be responsible). Nikon may include similar blockers in the si. The

Zeiss

LSM510 light path can be configured to use a microscope filter

cube. With

the right cube and a mirror slide, you can acquire the spectrum of

the Hg

lamp using the META, or you could buy a Lightform MIDL like Bob

Zucker's and

just lay it on the microscope. The META spectrum of the MIDL lamp

is not

even close to the calibration curve that comes with the MIDL. Bob

and Jeremy

have published the close match of the PARISS data for the MIDL.

Enjoy,

George

At 07:26 PM 10/5/2007, you wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks! We are interested in co-localization, for which we

sometimes

need the third dimension. Mainly we want to do spectral unmixing

for

multiple dyes and colocalization, so we really do need optical

slicing!

Thanks,

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig

you are correct.

Currently the PARISS only detects X-Y--not depth.

however, most data that I have seen from confocal spectral

systems are

only a single XY slice. they usually are not used to do a 3D

reconstruction in spectral mode.

bob

.

Robert M. Zucker, PhD

U.S. Environmental Protection Agency

Office of Research and Development

National Health and Environmental Effects Research Laboratory.

Telephone: 919-541-1585 Fax: 919-541-4017

e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67

2525 E.NC Highway 54

Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54

Durham, NC, 27713

   Craig Brideau

   <craig.brideau@G

   MAIL.COM>

To

   Sent by: [hidden email]

   Confocal

cc

   Microscopy List

   <CONFOCAL@LISTSE

   Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:44

   PM

Please respond

to

   Confocal

   Microscopy List

   <CONFOCAL@LISTSE

   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It doesn't seem to deal with depth though? It only detects in X-

Y, so

can it generate optical slices?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The PARISS system uses a Retiga 2000R cooled CCD camera. It is far

more

sensitive and accurate than any confocal microscope.

You can detect things in that system that can not be observed

with a

confocal spectral system. It is a great system in my opinion.

Bob

Robert M. Zucker, PhD

U.S. Environmental Protection Agency

Office of Research and Development

National Health and Environmental Effects Research Laboratory.

Telephone: 919-541-1585 Fax: 919-541-4017

e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67

2525 E.NC Highway 54

Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54

Durham, NC, 27713

   Craig Brideau

   <craig.brideau@G

   MAIL.COM>

To

   Sent by: [hidden email]

   Confocal

cc

   Microscopy List

   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 06:19

   PM

Please respond

to

   Confocal

   Microscopy List

   <CONFOCAL@LISTSE

   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Will this system have the same sort of sensitivity as a multi-anode

PMT system like the C1si? This PARISS system seems to use a CCD

camera as its detector, which will not be as sensitive, if I

understand correctly?

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Criag

If spectral analysis is the endpoint you may want to consider the

PARISS

system from Lightform. ( http://www.lightforminc.com/) that can be

placed on a widefield microscope.

We are using the PARISS system to obtain spectral data --it has

1nm

resolution and has a spectrum from 400-900nm. I find it to be a

great

asset in the laboratory,

It is far more sensitive and accurate that any confocal system

that

I

have seen.

Contact Jeremy Lerner for details on his system.

best wishes

Bob

LightForm, Inc.,

601 Route 206, Suite 26-479

   Hillsborough NJ 08844

   Tel: (908)281 9098

Email: [hidden email]

.

Robert M. Zucker, PhD

U.S. Environmental Protection Agency

Office of Research and Development

National Health and Environmental Effects Research Laboratory.

Telephone: 919-541-1585 Fax: 919-541-4017

e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67

2525 E.NC Highway 54

Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54

Durham, NC, 27713

   Craig Brideau

   <craig.brideau@G

   MAIL.COM>

To

   Sent by:

[hidden email]

   Confocal

cc

   Microscopy List

   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/05/2007 05:48

   PM

Please respond

to

   Confocal

   Microscopy List

   <CONFOCAL@LISTSE

   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thanks for your input! As in everything, there's a trade-off.

Still,

for us the spectral data will be worth it.

Craig

On 10/5/07, Robert Zucker <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig

The efficiency of detection is much less in the spectral mode due

to

the

design of the multianode detector. In addition as Kurt Thorn said

the

light is split into a number of different bandpass channels

instead

of

being detected by only one channel which also limits the

detection

in

each channel.

Nikon compensates for this decreased light by increasing the

pixel

dwell

time. However photons are photons. With less photons you will get

a

noisier image.

The spectral detection of any confocal system using a multianode

detector will not be as good as a PMT designed for good

sensitivity

and

low light detection. It will produce nosier images but if

there is

enough light you will be able to determine a valuable spectrum

which

can

be used for scientific experiments and to manipulate images. Like

everything with confocal microscopy there is trade-offs and no

perfect

system.

Best wishes.

Bob

Robert M. Zucker, PhD

U.S. Environmental Protection Agency

Office of Research and Development

National Health and Environmental Effects Research Laboratory.

Telephone: 919-541-1585 Fax: 919-541-4017

e-mail: [hidden email]

Mail address: Reproductive Toxicology Division, MD 67

2525 E.NC Highway 54

Research Triangle Park, North Carolina, 27711

Shipping address: 2525 E.NC Highway 54

Durham, NC, 27713

   Craig Brideau

   <craig.brideau@G

   MAIL.COM>

To

   Sent by:

[hidden email]

   Confocal

cc

   Microscopy List

   <CONFOCAL@LISTSE

Subject

   RV.BUFFALO.EDU> Re: Nikon C1si?

   10/04/2007 02:49

   PM

Please respond

to

   Confocal

   Microscopy List

   <CONFOCAL@LISTSE

   RV.BUFFALO.EDU>

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I was wondering if you saw this decreased throughput in spectral

mode

only, or also in conventional detection mode? The spectral mode

works

with a multi-anode PMT, which has a lower quantum efficiency than

a

conventional PMT. From what I have seen of the design, it also

has

the option to use a conventional PMT system with the device,

and I

am

wondering if you have also tried that mode of operation?

Thanks,

Craig

On 10/4/07, Robert Zucker <[hidden email]> wrote:

One of the major limitations that we have seen with this unit

and

also

with the Zeiss meta 510 is the decreased light throughput .

This

creates images that are noisier than conventional confocal

microscopes.

In our hands it appears you will need to have a bright sample to

make

the spectral system work properly. ,

George McNamara, Ph.D.

University of Miami, Miller School of Medicine

Image Core

Miami, FL 33010

[hidden email]

[hidden email]

305-243-8436 office

http://home.earthlink.net/~pubspectra/

http://home.earthlink.net/~geomcnamara/

http://www.sylvester.org/health_pro/shared_resources/index.asp

(see Analytical Imaging Core Facility)

George McNamara

Re: Nikon C1si? -spectral unmixing

In reply to this post by Judy Trogadis

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Judy,

Fluorescence Imaging Spectroscopy and Microscopy (Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications) by X. F. Wang and Brian Herman (Paperback - April 1996), especially Yuval Garini's chapter.

Thumb through the articles in the August 2006 issue of Cytometry (volume 69 number 8) and look at the figures. The Thaler and Vogel article is especially nice.

Tsurui et al 2003 J Histochem Cytochem 48: 653-662 (www.jhc.org), though I would try to avoid acquiring 3 data sets for 7 fluorophores.

Schrock et al 1996 Science 273: 494-497, especially the explanatory figure. By the way, there are now over 580 SKY (spectral karyotyp*) papers in PubMed since this paper (ok, some are reviews, but not as high a ratio reviews:research as for FRET microscopy), which is double the number of spectral confocal papers (329, but 64 are Raman).

Two papers that should be read as a pair are Garini 1999 Cytometry 35: 214-226 and Castleman 2000 41: 139-147.

George

At 11:59 AM 10/8/2007, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

In light of this discussion, can anyone suggest some recent practical references on spectral unmixing?

Thanks.
Judy

Judy Trogadis
Bio-Imaging Coordinator
St. Michael's Hospital, 7Queen
30 Bond St.
Toronto, ON M5B 1W8, Canada
ph: 416-864-6060 x6337
pager: 416-685-9219
fax: 416-864-6043
[hidden email]

>>> Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

You make some interesting points! It does seem that the low threshold
capability of PMT's make them more useful for our photon counting type
experiments. Still, in the case of a brighter sample the speed of an
EMCCD would be useful.

Thanks,

Craig

On 10/6/07, George McNamara <[hidden email]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Hi Craig,
>
> The Lightform PARISS has a detection slit in front of the spectral
> dispersion element and the CCD. This makes it half-confocal. If a matching
> slit is placed in the epi-illumination field aperture plane (or a conjugate
> plane) it is a slit confocal (aka line scan confocal). The result is an
> X*Spectral image. If the microscope has a motorized stage, the Y-axis can be
> added = push broom confocal imaging spectrometer. The performance is such
> that the emission filter can be removed from the epi-illumination filter
> cube when used with an Hg lamp (the bright Hg line or lines are one
> nanometer or so side = 1 or a few pixels, so do not bleed into the emission
> areas and can be used to quantify the excitation power). The PARISS can also
> be used for brightfield images.
>
> You can contact Jeremy Lerner at http://www.lightforminc.com/ for
> additional details about the PARISS.
>
>
> With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point
> scanning confocal microscope detects a maximum of about 8 photons per pixel
> per scan. This is a nice match to a PMT (or APD for far red and infrared
> photons), not so good for a CCD (see Jim's post about noise). On the other
> hand, a CCD is an area detector (or line * spectrum detector for the
> PARISS), so the CCD is a great way to acquire 2D images fast. A CCD or EMCCD
> system can be used to image a thin optical section, quickly, with judicious
> use of excitation light, as in TIRF, fast multipoint multiphoton scanners
> such as the TriMScope (if it can be made to work reliably) or Scherer's
> stochastic scanner (
> http://www.opticsexpress.org/abstract.cfm?id=89328), or
> hopefully by widefield multiphoton excitation (Brooker 2007 US patent
> 7,170,675 ), or by multiplane acquisition followed by digital deconvolution.
> If you can buy or borrow 3 EMCCD's like Ian Parker, you can acquire multiple
> focal planes simultaneously, see PubMed 17716727, also similar rig by
> Prabhat, PubMed 17384151.
>
> Back to 32-channel PMT confocal microscopes: the Zeiss META detector
> includes blockers for the major laser lines. As far as I am aware, they are
> in the light path all the time (the 488 nm blocker may be responsible for
> zigzag artifacts in CFP emission spectra, though bad PMT channels could also
> be responsible). Nikon may include similar blockers in the si. The Zeiss
> LSM510 light path can be configured to use a microscope filter cube. With
> the right cube and a mirror slide, you can acquire the spectrum of the Hg
> lamp using the META, or you could buy a Lightform MIDL like Bob Zucker's and
> just lay it on the microscope. The META spectrum of the MIDL lamp is not
> even close to the calibration curve that comes with the MIDL. Bob and Jeremy
> have published the close match of the PARISS data for the MIDL.
>
>
> Enjoy,
>
>
> George
>
>
>
> At 07:26 PM 10/5/2007, you wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Thanks! We are interested in co-localization, for which we sometimes
> need the third dimension. Mainly we want to do spectral unmixing for
> multiple dyes and colocalization, so we really do need optical
> slicing!
>
> Thanks,
>
> Craig
>
>
> On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Craig
> > you are correct.
> > Currently the PARISS only detects X-Y--not depth.
> > however, most data that I have seen from confocal spectral systems are
> > only a single XY slice. they usually are not used to do a 3D
> > reconstruction in spectral mode.
> > bob
> > .
> >
> >
> > Robert M. Zucker, PhD
> > U.S. Environmental Protection Agency
> > Office of Research and Development
> > National Health and Environmental Effects Research Laboratory.
> > Telephone: 919-541-1585   Fax: 919-541-4017
> > e-mail: [hidden email]
> >
> > Mail address: Reproductive Toxicology Division, MD 67
> > 2525 E.NC Highway 54
> > Research Triangle Park, North Carolina, 27711
> >
> > Shipping address: 2525 E.NC Highway 54
> > Durham, NC, 27713
> >
> >
> >
> >
> >              Craig Brideau
> >              <craig.brideau@G
> >              MAIL.COM>
>             To
> >              Sent by:                 [hidden email]
> >              Confocal
>             cc
> >              Microscopy List
> >              <CONFOCAL@LISTSE
>      Subject
> >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> >
> >
> >              10/05/2007 06:44
> >              PM
> >
> >
> >               Please respond
> >                     to
> >                  Confocal
> >              Microscopy List
> >              <CONFOCAL@LISTSE
> >              RV.BUFFALO.EDU>
> >
> >
> >
> >
> >
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > It doesn't seem to deal with depth though? It only detects in X-Y, so
> > can it generate optical slices?
> >
> > Craig
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> > more
> > > sensitive and accurate than any confocal microscope.
> > > You can detect things in that system that can not be observed with a
> > > confocal spectral system. It is a great system in my opinion.
> > > Bob
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585   Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > >              Craig Brideau
> > >              <craig.brideau@G
> > >              MAIL.COM>
> > To
> > >              Sent by:                 [hidden email]
> > >              Confocal
> > cc
> > >              Microscopy List
> > >              <CONFOCAL@LISTSE
> > Subject
> > >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> > >
> > >
> > >              10/05/2007 06:19
> > >              PM
> > >
> > >
> > >               Please respond
> > >                     to
> > >                  Confocal
> > >              Microscopy List
> > >              <CONFOCAL@LISTSE
> > >              RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Will this system have the same sort of sensitivity as a multi-anode
> > > PMT system like the C1si? This PARISS system seems to use a CCD
> > > camera as its detector, which will not be as sensitive, if I
> > > understand correctly?
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Criag
> > > > If spectral analysis is the endpoint you may want to consider the
> > > PARISS
> > > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > > placed on a widefield microscope.
> > > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > > resolution and has a spectrum from 400-900nm. I find it to be a
> > great
> > > > asset in the laboratory,
> > > > It is far more sensitive and accurate that any confocal system that
> > I
> > > > have seen.
> > > >
> > > > Contact Jeremy Lerner for details on his system.
> > > > best wishes
> > > > Bob
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >                                 LightForm, Inc.,
> > > >                           601 Route 206, Suite 26-479
> > > >                              Hillsborough NJ 08844
> > > >                                Tel: (908)281 9098
> > > >                         Email: [hidden email]
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > .
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585   Fax: 919-541-4017
> > > > e-mail: [hidden email]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > >              Craig Brideau
> > > >              <craig.brideau@G
> > > >              MAIL.COM>
> > > To
> > > >              Sent by:                 [hidden email]
> > > >              Confocal
> > > cc
> > > >              Microscopy List
> > > >              <CONFOCAL@LISTSE
> > > Subject
> > > >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> > > >
> > > >
> > > >              10/05/2007 05:48
> > > >              PM
> > > >
> > > >
> > > >               Please respond
> > > >                     to
> > > >                  Confocal
> > > >              Microscopy List
> > > >              <CONFOCAL@LISTSE
> > > >              RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Thanks for your input! As in everything, there's a trade-off.
> > Still,
> > > > for us the spectral data will be worth it.
> > > >
> > > > Craig
> > > >
> > > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > Craig
> > > > > The efficiency of detection is much less in the spectral mode due
> > to
> > > > the
> > > > > design of the multianode detector. In addition as Kurt Thorn said
> > > the
> > > > > light is split into a number of different bandpass channels
> > instead
> > > of
> > > > > being detected by only one channel which also limits the detection
> > > in
> > > > > each channel.
> > > > > Nikon compensates for this decreased light by increasing the pixel
> > > > dwell
> > > > > time. However photons are photons. With less photons you will get
> > a
> > > > > noisier image.
> > > > > The spectral detection of any confocal system using a multianode
> > > > > detector will not be as good as a PMT designed for good
> > sensitivity
> > > > and
> > > > > low light detection. It will produce nosier images but if there is
> > > > > enough light you will be able to determine a valuable spectrum
> > which
> > > > can
> > > > > be used for scientific experiments and to manipulate images. Like
> > > > > everything with confocal microscopy there is trade-offs and no
> > > perfect
> > > > > system.
> > > > > Best wishes.
> > > > > Bob
> > > > >
> > > > > Robert M. Zucker, PhD
> > > > > U.S. Environmental Protection Agency
> > > > > Office of Research and Development
> > > > > National Health and Environmental Effects Research Laboratory.
> > > > > Telephone: 919-541-1585   Fax: 919-541-4017
> > > > > e-mail: [hidden email]
> > > > >
> > > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > > 2525 E.NC Highway 54
> > > > > Research Triangle Park, North Carolina, 27711
> > > > >
> > > > > Shipping address: 2525 E.NC Highway 54
> > > > > Durham, NC, 27713
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >              Craig Brideau
> > > > >              <craig.brideau@G
> > > > >              MAIL.COM>
> > > > To
> > > > >              Sent by:
> > [hidden email]
> > > > >              Confocal
> > > > cc
> > > > >              Microscopy List
> > > > >              <CONFOCAL@LISTSE
> > > > Subject
> > > > >              RV.BUFFALO.EDU>          Re: Nikon C1si?
> > > > >
> > > > >
> > > > >              10/04/2007 02:49
> > > > >              PM
> > > > >
> > > > >
> > > > >               Please respond
> > > > >                     to
> > > > >                  Confocal
> > > > >              Microscopy List
> > > > >              <CONFOCAL@LISTSE
> > > > >              RV.BUFFALO.EDU>
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Search the CONFOCAL archive at
> > > > >
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > I was wondering if you saw this decreased throughput in spectral
> > > mode
> > > > > only, or also in conventional detection mode? The spectral mode
> > > works
> > > > > with a multi-anode PMT, which has a lower quantum efficiency than
> > a
> > > > > conventional PMT. From what I have seen of the design, it also
> > has
> > > > > the option to use a conventional PMT system with the device, and I
> > > am
> > > > > wondering if you have also tried that mode of operation?
> > > > >
> > > > > Thanks,
> > > > >
> > > > > Craig
> > > > >
> > > > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > > > One of the major limitations that we have seen with this unit
> > and
> > > > also
> > > > > > with the Zeiss meta 510 is the decreased light throughput .
> > This
> > > > > > creates images that are noisier than conventional confocal
> > > > > microscopes.
> > > > > > In our hands it appears you will need to have a bright sample to
> > > > make
> > > > > > the spectral system work properly. ,
> > > > >
> > > >
> > >
> >
>
>
>
>
>
>
> George McNamara, Ph.D.
> University of Miami, Miller School of Medicine
> Image Core
> Miami, FL 33010
> [hidden email]
> [hidden email]
> 305-243-8436 office
> http://home.earthlink.net/~pubspectra/
> http://home.earthlink.net/~geomcnamara/
> http://www.sylvester.org/health_pro/shared_resources/index.asp
> (see Analytical Imaging Core Facility)
>
>
>

Judy Trogadis

Re: Nikon C1si? -spectral unmixing

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for all the references.
Judy

>>> George McNamara <[hidden email]> 10/09/07 8:12 PM >>>
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Judy,

<http://www.amazon.com/Fluorescence-Spectroscopy-Microscopy-Chemical-Analysis/dp/047101527X/ref=sr_1_1/102-0290541-1997704?ie=UTF8&s=books&qid=1191974379&sr=1-1>Fluorescence
Imaging Spectroscopy and Microscopy (Chemical Analysis: A Series of
Monographs on Analytical Chemistry and Its Applications) by X. F.
Wang and Brian Herman (Paperback - April 1996), especially Yuval
Garini's chapter.

Thumb through the articles in the August 2006 issue of Cytometry
(volume 69 number 8) and look at the figures. The Thaler and Vogel
article is especially nice.

Tsurui et al 2003 J Histochem Cytochem 48: 653-662 (www.jhc.org),
though I would try to avoid acquiring 3 data sets for 7 fluorophores.

Schrock et al 1996 Science 273: 494-497, especially the explanatory
figure. By the way, there are now over 580 SKY (spectral karyotyp*)
papers in PubMed since this paper (ok, some are reviews, but not as
high a ratio reviews:research as for FRET microscopy), which is
double the number of spectral confocal papers (329, but 64 are Raman).

Two papers that should be read as a pair are Garini 1999 Cytometry
35: 214-226 and Castleman 2000 41: 139-147.

George

At 11:59 AM 10/8/2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>In light of this discussion, can anyone suggest some recent
>practical references on spectral unmixing?
>
>Thanks.
>Judy
>
>
>
>Judy Trogadis
>Bio-Imaging Coordinator
>St. Michael's Hospital, 7Queen
>30 Bond St.
>Toronto, ON M5B 1W8, Canada
>ph: 416-864-6060 x6337
>pager: 416-685-9219
>fax: 416-864-6043
>[hidden email]
>
>
> >>> Craig Brideau <[hidden email]> 10/6/2007 5:31 PM >>>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>You make some interesting points! It does seem that the low threshold
>capability of PMT's make them more useful for our photon counting type
>experiments. Still, in the case of a brighter sample the speed of an
>EMCCD would be useful.
>
>Thanks,
>
>Craig
>
>On 10/6/07, George McNamara <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > Hi Craig,
> >
> > The Lightform PARISS has a detection slit in front of the spectral
> > dispersion element and the CCD. This makes it half-confocal. If a matching
> > slit is placed in the epi-illumination field aperture plane (or a conjugate
> > plane) it is a slit confocal (aka line scan confocal). The result is an
> > X*Spectral image. If the microscope has a motorized stage, the
> Y-axis can be
> > added = push broom confocal imaging spectrometer. The performance is such
> > that the emission filter can be removed from the epi-illumination filter
> > cube when used with an Hg lamp (the bright Hg line or lines are one
> > nanometer or so side = 1 or a few pixels, so do not bleed into the emission
> > areas and can be used to quantify the excitation power). The
> PARISS can also
> > be used for brightfield images.
> >
> > You can contact Jeremy Lerner at http://www.lightforminc.com/ for
> > additional details about the PARISS.
> >
> >
> > With respect to PMT vs CCD or EMCCD, Pawley's rule is that a typical point
> > scanning confocal microscope detects a maximum of about 8 photons per pixel
> > per scan. This is a nice match to a PMT (or APD for far red and infrared
> > photons), not so good for a CCD (see Jim's post about noise). On the other
> > hand, a CCD is an area detector (or line * spectrum detector for the
> > PARISS), so the CCD is a great way to acquire 2D images fast. A
> CCD or EMCCD
> > system can be used to image a thin optical section, quickly, with judicious
> > use of excitation light, as in TIRF, fast multipoint multiphoton scanners
> > such as the TriMScope (if it can be made to work reliably) or Scherer's
> > stochastic scanner (
> > http://www.opticsexpress.org/abstract.cfm?id=89328), or
> > hopefully by widefield multiphoton excitation (Brooker 2007 US patent
> > 7,170,675 ), or by multiplane acquisition followed by digital
> deconvolution.
> > If you can buy or borrow 3 EMCCD's like Ian Parker, you can
> acquire multiple
> > focal planes simultaneously, see PubMed 17716727, also similar rig by
> > Prabhat, PubMed 17384151.
> >
> > Back to 32-channel PMT confocal microscopes: the Zeiss META detector
> > includes blockers for the major laser lines. As far as I am aware, they are
> > in the light path all the time (the 488 nm blocker may be responsible for
> > zigzag artifacts in CFP emission spectra, though bad PMT channels
> could also
> > be responsible). Nikon may include similar blockers in the si. The Zeiss
> > LSM510 light path can be configured to use a microscope filter cube. With
> > the right cube and a mirror slide, you can acquire the spectrum of the Hg
> > lamp using the META, or you could buy a Lightform MIDL like Bob
> Zucker's and
> > just lay it on the microscope. The META spectrum of the MIDL lamp is not
> > even close to the calibration curve that comes with the MIDL. Bob
> and Jeremy
> > have published the close match of the PARISS data for the MIDL.
> >
> >
> > Enjoy,
> >
> >
> > George
> >
> >
> >
> > At 07:26 PM 10/5/2007, you wrote:
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Thanks! We are interested in co-localization, for which we sometimes
> > need the third dimension. Mainly we want to do spectral unmixing for
> > multiple dyes and colocalization, so we really do need optical
> > slicing!
> >
> > Thanks,
> >
> > Craig
> >
> >
> > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Craig
> > > you are correct.
> > > Currently the PARISS only detects X-Y--not depth.
> > > however, most data that I have seen from confocal spectral systems are
> > > only a single XY slice. they usually are not used to do a 3D
> > > reconstruction in spectral mode.
> > > bob
> > > .
> > >
> > >
> > > Robert M. Zucker, PhD
> > > U.S. Environmental Protection Agency
> > > Office of Research and Development
> > > National Health and Environmental Effects Research Laboratory.
> > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > e-mail: [hidden email]
> > >
> > > Mail address: Reproductive Toxicology Division, MD 67
> > > 2525 E.NC Highway 54
> > > Research Triangle Park, North Carolina, 27711
> > >
> > > Shipping address: 2525 E.NC Highway 54
> > > Durham, NC, 27713
> > >
> > >
> > >
> > >
> > > Craig Brideau
> > > <craig.brideau@G
> > > MAIL.COM>
> > To
> > > Sent by: [hidden email]
> > > Confocal
> > cc
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > Subject
> > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > >
> > >
> > > 10/05/2007 06:44
> > > PM
> > >
> > >
> > > Please respond
> > > to
> > > Confocal
> > > Microscopy List
> > > <CONFOCAL@LISTSE
> > > RV.BUFFALO.EDU>
> > >
> > >
> > >
> > >
> > >
> > >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > It doesn't seem to deal with depth though? It only detects in X-Y, so
> > > can it generate optical slices?
> > >
> > > Craig
> > >
> > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > Search the CONFOCAL archive at
> > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > The PARISS system uses a Retiga 2000R cooled CCD camera. It is far
> > > more
> > > > sensitive and accurate than any confocal microscope.
> > > > You can detect things in that system that can not be observed with a
> > > > confocal spectral system. It is a great system in my opinion.
> > > > Bob
> > > >
> > > > Robert M. Zucker, PhD
> > > > U.S. Environmental Protection Agency
> > > > Office of Research and Development
> > > > National Health and Environmental Effects Research Laboratory.
> > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > e-mail: [hidden email]
> > > >
> > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > 2525 E.NC Highway 54
> > > > Research Triangle Park, North Carolina, 27711
> > > >
> > > > Shipping address: 2525 E.NC Highway 54
> > > > Durham, NC, 27713
> > > >
> > > >
> > > >
> > > >
> > > > Craig Brideau
> > > > <craig.brideau@G
> > > > MAIL.COM>
> > > To
> > > > Sent by: [hidden email]
> > > > Confocal
> > > cc
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > Subject
> > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > >
> > > >
> > > > 10/05/2007 06:19
> > > > PM
> > > >
> > > >
> > > > Please respond
> > > > to
> > > > Confocal
> > > > Microscopy List
> > > > <CONFOCAL@LISTSE
> > > > RV.BUFFALO.EDU>
> > > >
> > > >
> > > >
> > > >
> > > >
> > > >
> > > > Search the CONFOCAL archive at
> > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > >
> > > > Will this system have the same sort of sensitivity as a multi-anode
> > > > PMT system like the C1si? This PARISS system seems to use a CCD
> > > > camera as its detector, which will not be as sensitive, if I
> > > > understand correctly?
> > > >
> > > > Craig
> > > >
> > > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > > Search the CONFOCAL archive at
> > > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > Criag
> > > > > If spectral analysis is the endpoint you may want to consider the
> > > > PARISS
> > > > > system from Lightform. ( http://www.lightforminc.com/) that can be
> > > > > placed on a widefield microscope.
> > > > > We are using the PARISS system to obtain spectral data --it has 1nm
> > > > > resolution and has a spectrum from 400-900nm. I find it to be a
> > > great
> > > > > asset in the laboratory,
> > > > > It is far more sensitive and accurate that any confocal system that
> > > I
> > > > > have seen.
> > > > >
> > > > > Contact Jeremy Lerner for details on his system.
> > > > > best wishes
> > > > > Bob
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > LightForm, Inc.,
> > > > > 601 Route 206, Suite 26-479
> > > > > Hillsborough NJ 08844
> > > > > Tel: (908)281 9098
> > > > > Email: [hidden email]
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > .
> > > > >
> > > > > Robert M. Zucker, PhD
> > > > > U.S. Environmental Protection Agency
> > > > > Office of Research and Development
> > > > > National Health and Environmental Effects Research Laboratory.
> > > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > > e-mail: [hidden email]
> > > > >
> > > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > > 2525 E.NC Highway 54
> > > > > Research Triangle Park, North Carolina, 27711
> > > > >
> > > > > Shipping address: 2525 E.NC Highway 54
> > > > > Durham, NC, 27713
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Craig Brideau
> > > > > <craig.brideau@G
> > > > > MAIL.COM>
> > > > To
> > > > > Sent by: [hidden email]
> > > > > Confocal
> > > > cc
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > Subject
> > > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > > >
> > > > >
> > > > > 10/05/2007 05:48
> > > > > PM
> > > > >
> > > > >
> > > > > Please respond
> > > > > to
> > > > > Confocal
> > > > > Microscopy List
> > > > > <CONFOCAL@LISTSE
> > > > > RV.BUFFALO.EDU>
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > >
> > > > > Search the CONFOCAL archive at
> > > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > >
> > > > > Thanks for your input! As in everything, there's a trade-off.
> > > Still,
> > > > > for us the spectral data will be worth it.
> > > > >
> > > > > Craig
> > > > >
> > > > > On 10/5/07, Robert Zucker <[hidden email]> wrote:
> > > > > > Search the CONFOCAL archive at
> > > > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > > >
> > > > > > Craig
> > > > > > The efficiency of detection is much less in the spectral mode due
> > > to
> > > > > the
> > > > > > design of the multianode detector. In addition as Kurt Thorn said
> > > > the
> > > > > > light is split into a number of different bandpass channels
> > > instead
> > > > of
> > > > > > being detected by only one channel which also limits the detection
> > > > in
> > > > > > each channel.
> > > > > > Nikon compensates for this decreased light by increasing the pixel
> > > > > dwell
> > > > > > time. However photons are photons. With less photons you will get
> > > a
> > > > > > noisier image.
> > > > > > The spectral detection of any confocal system using a multianode
> > > > > > detector will not be as good as a PMT designed for good
> > > sensitivity
> > > > > and
> > > > > > low light detection. It will produce nosier images but if there is
> > > > > > enough light you will be able to determine a valuable spectrum
> > > which
> > > > > can
> > > > > > be used for scientific experiments and to manipulate images. Like
> > > > > > everything with confocal microscopy there is trade-offs and no
> > > > perfect
> > > > > > system.
> > > > > > Best wishes.
> > > > > > Bob
> > > > > >
> > > > > > Robert M. Zucker, PhD
> > > > > > U.S. Environmental Protection Agency
> > > > > > Office of Research and Development
> > > > > > National Health and Environmental Effects Research Laboratory.
> > > > > > Telephone: 919-541-1585 Fax: 919-541-4017
> > > > > > e-mail: [hidden email]
> > > > > >
> > > > > > Mail address: Reproductive Toxicology Division, MD 67
> > > > > > 2525 E.NC Highway 54
> > > > > > Research Triangle Park, North Carolina, 27711
> > > > > >
> > > > > > Shipping address: 2525 E.NC Highway 54
> > > > > > Durham, NC, 27713
> > > > > >
> > > > > >
> > > > > >
> > > > > >
> > > > > > Craig Brideau
> > > > > > <craig.brideau@G
> > > > > > MAIL.COM>
> > > > > To
> > > > > > Sent by:
> > > [hidden email]
> > > > > > Confocal
> > > > > cc
> > > > > > Microscopy List
> > > > > > <CONFOCAL@LISTSE
> > > > > Subject
> > > > > > RV.BUFFALO.EDU> Re: Nikon C1si?
> > > > > >
> > > > > >
> > > > > > 10/04/2007 02:49
> > > > > > PM
> > > > > >
> > > > > >
> > > > > > Please respond
> > > > > > to
> > > > > > Confocal
> > > > > > Microscopy List
> > > > > > <CONFOCAL@LISTSE
> > > > > > RV.BUFFALO.EDU>
> > > > > >
> > > > > >
> > > > > >
> > > > > >
> > > > > >
> > > > > >
> > > > > > Search the CONFOCAL archive at
> > > > > >
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > > > > >
> > > > > > I was wondering if you saw this decreased throughput in spectral
> > > > mode
> > > > > > only, or also in conventional detection mode? The spectral mode
> > > > works
> > > > > > with a multi-anode PMT, which has a lower quantum efficiency than
> > > a
> > > > > > conventional PMT. From what I have seen of the design, it also
> > > has
> > > > > > the option to use a conventional PMT system with the device, and I
> > > > am
> > > > > > wondering if you have also tried that mode of operation?
> > > > > >
> > > > > > Thanks,
> > > > > >
> > > > > > Craig
> > > > > >
> > > > > > On 10/4/07, Robert Zucker <[hidden email]> wrote:
> > > > > > > One of the major limitations that we have seen with this unit
> > > and
> > > > > also
> > > > > > > with the Zeiss meta 510 is the decreased light throughput .
> > > This
> > > > > > > creates images that are noisier than conventional confocal
> > > > > > microscopes.
> > > > > > > In our hands it appears you will need to have a bright sample to
> > > > > make
> > > > > > > the spectral system work properly. ,
> > > > > >
> > > > >
> > > >
> > >
> >
> >
> >
> >
> >
> >
> > George McNamara, Ph.D.
> > University of Miami, Miller School of Medicine
> > Image Core
> > Miami, FL 33010
> > [hidden email]
> > [hidden email]
> > 305-243-8436 office
> > http://home.earthlink.net/~pubspectra/
> > http://home.earthlink.net/~geomcnamara/
> > http://www.sylvester.org/health_pro/shared_resources/index.asp
> > (see Analytical Imaging Core Facility)
> >
> >
> >