Re: Pulsed LED light sources for less photobleaching - was: Guidance wanted on illumination stability

[Daniel James White]

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Hi Jeremy and David,

On Dec 1, 2010, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> On Nov 30, 2010, at 10:26 AM, Jeremy Adler wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>> *****
>> =20
>> It might also be useful to have pulsatile LEDs operating in the =20
>> nanosecond range - there was a Stefan Hell paper that showed very =20
>> significant reductions in photobleaching when the gap between pulses =20=

I am very interested in this, applied to wide field illumination.

>
> Date:    Tue, 30 Nov 2010 11:19:40 -0500
> From:    David Knecht <[hidden email]>
> Subject: Re: Guidance wanted on illumination stability
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> It is not only possible, but Thorlabs sells an LED controller that =
> modulates LED intensity in this way up to 10kHz which I think should be =
> adequate for the effect discussed in the Hell paper.  I have not had a =
> chance to try it yet and see if it works to reduce bleaching.  Dave
> http://www.thorlabs.com/NewGroupPage9.cfm?ObjectGroup_ID=3D4003

that link doesnt seem to work....

or is it this?

http://www.thorlabs.de/NewGroupPage9.cfm?ObjectGroup_ID=3459

in any case, I'm not sure it s quite the right thing to avoid the bleaching and photoxicity:

What we need is 10 ns ish pulses separated by period of zero illumination separated by 10s of microseconds...

for example
http://www.rapp-opto.com/index.php?show=products&id=38

.. over simplified explanation below

Considering the photophysics of say GFP...

1) the fluorescence lifetime is a few ns,
so intense pulses in the low ns range duration could put nearly all the GFPs into excited fluorescent state....
but with not too much double excitation (which might be a bad thing - bleaching etc. )

2) Next the GFP excited state relaxes in different possible ways
        a) fluorescence emission (what we want) after a a few ns
        b) non radiative energy loss (dark)
        c) inter system crossing to the triplet state
                This is bad news since:
                        i) it is effectively dark (in the fluo emission waveband)
                        ii) it is very long lived compared to the single excited state... T1 lifetime is in the region of 10  micro seconds.

This means that, in the typical widefield continuous wave illumination, especially at very very high illumination power,
the triplet state is rapidly populated and builds up up in concentration over the exposure time of 10 to 100 to 1000s of milliseconds of a CCD camera.

Potentially, much of the dye could be "trapped"  in a dark triplet state after the first few milliseconds of exposure.
And there can be multiple excitation into higher excited states... also bad news for phototoxicity and bleaching.

Some (including me) seem to think that this is why you get more signal than you think you should on a fast scanning point scanner or spinning disk
which give each point in the sample effectively pulsed illumination,
and also that its the the way to prevent bleaching and phototoxicity:

I mean one could use:
1) excitation pulses that are about the same duration as the fluorescence lifetime (or should it be the fluorescence excitation time... but thats very short... depends on the power)
2|) separation between the pulses of no excitation light power which are a bit longer than the triplet state lifetime...
giving it chance to relax before it gets hit with another excitation photon that would bleach it and cause production of reactive oxygen species and other nastiness.

So a square wave shape is needed with very long gaps compared to the very short pulses. ....
and the modulated LED doesn't do that really... its a sine wave right?
Or am I totally wrong?

cheers

Dan



Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Craig Brideau]

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Sounds like you need duty-cycle control over the square wave.  Basically
have a short 'on' time with a long 'off' time to let the dye relax, then hit
it again.  The problem is, can the electronics give you the appropriately
short 'on' you need?
I'd say a square wave is the way to go, since you need specific on and off
times.

Craig


On Wed, Dec 1, 2010 at 2:06 AM, Daniel James White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Jeremy and David,
>
> On Dec 1, 2010, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system
> wrote:
>
> > On Nov 30, 2010, at 10:26 AM, Jeremy Adler wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> >> *****
> >> =20
> >> It might also be useful to have pulsatile LEDs operating in the =20
> >> nanosecond range - there was a Stefan Hell paper that showed very =20
> >> significant reductions in photobleaching when the gap between pulses
> =20=
>
> I am very interested in this, applied to wide field illumination.
>
> >
> > Date:    Tue, 30 Nov 2010 11:19:40 -0500
> > From:    David Knecht <[hidden email]>
> > Subject: Re: Guidance wanted on illumination stability
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> > *****
> >
> > It is not only possible, but Thorlabs sells an LED controller that =
> > modulates LED intensity in this way up to 10kHz which I think should be =
> > adequate for the effect discussed in the Hell paper.  I have not had a =
> > chance to try it yet and see if it works to reduce bleaching.  Dave
> > http://www.thorlabs.com/NewGroupPage9.cfm?ObjectGroup_ID=3D4003
>
> that link doesnt seem to work....
>
> or is it this?
>
> http://www.thorlabs.de/NewGroupPage9.cfm?ObjectGroup_ID=3459
>
> in any case, I'm not sure it s quite the right thing to avoid the bleaching
> and photoxicity:
>
> What we need is 10 ns ish pulses separated by period of zero illumination
> separated by 10s of microseconds...
>
> for example
> http://www.rapp-opto.com/index.php?show=products&id=38
>
> .. over simplified explanation below
>
> Considering the photophysics of say GFP...
>
> 1) the fluorescence lifetime is a few ns,
> so intense pulses in the low ns range duration could put nearly all the
> GFPs into excited fluorescent state....
> but with not too much double excitation (which might be a bad thing -
> bleaching etc. )
>
> 2) Next the GFP excited state relaxes in different possible ways
>        a) fluorescence emission (what we want) after a a few ns
>        b) non radiative energy loss (dark)
>        c) inter system crossing to the triplet state
>                This is bad news since:
>                        i) it is effectively dark (in the fluo emission
> waveband)
>                        ii) it is very long lived compared to the single
> excited state... T1 lifetime is in the region of 10  micro seconds.
>
> This means that, in the typical widefield continuous wave illumination,
> especially at very very high illumination power,
> the triplet state is rapidly populated and builds up up in concentration
> over the exposure time of 10 to 100 to 1000s of milliseconds of a CCD
> camera.
>
> Potentially, much of the dye could be "trapped"  in a dark triplet state
> after the first few milliseconds of exposure.
> And there can be multiple excitation into higher excited states... also bad
> news for phototoxicity and bleaching.
>
> Some (including me) seem to think that this is why you get more signal than
> you think you should on a fast scanning point scanner or spinning disk
> which give each point in the sample effectively pulsed illumination,
> and also that its the the way to prevent bleaching and phototoxicity:
>
> I mean one could use:
> 1) excitation pulses that are about the same duration as the fluorescence
> lifetime (or should it be the fluorescence excitation time... but thats very
> short... depends on the power)
> 2|) separation between the pulses of no excitation light power which are a
> bit longer than the triplet state lifetime...
> giving it chance to relax before it gets hit with another excitation photon
> that would bleach it and cause production of reactive oxygen species and
> other nastiness.
>
> So a square wave shape is needed with very long gaps compared to the very
> short pulses. ....
> and the modulated LED doesn't do that really... its a sine wave right?
> Or am I totally wrong?
>
> cheers
>
> Dan
>
>
>
> Dr. Daniel James White BSc. (Hons.) PhD
> Senior Microscopist / Image Visualisation, Processing and Analysis
> Light Microscopy and Image Processing Facilities
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
>
> http://www.bioimagexd.net       BioImageXD
> http://pacific.mpi-cbg.de               Fiji -  is just ImageJ (Batteries
> Included)
> http://www.chalkie.org.uk               Dan's Homepages
> https://ifn.mpi-cbg.de                  Dresden Imaging Facility Network
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>

[James Pawley]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Dan

This matter of "pulsed vs. continuous" excitation
seems to be fraught with many uncertainties.

How do we insure that we get the same number of
excitation photons striking a fixed area of the
specimen in the two modes? Do we just use a CCD
camera set up to record for a fixed time period,
adjust the continuous excitation level so that it
produces the same signal level as we get from a
pulsed illumination source, and then compare the
amount of bleaching produced in each case? How
are we sure that the excitation spectra are the
same? We know the spectrum of a laser pretty well
but an LED spectrum multiplied by the effects of
an excitation filter and beamsplitter may not
look the same as that which gets through these
same filters from an Hg arc (which is very
"spikey" in the visible). Much of the work on
pulsed illumination so far has been done with
2-photon lasers which introduce a whole raft of
additional unknowns (shape of pulse in time and
space, 2-photon cross-section, 2-photon damage
mechanisms...)

As for the photophysics, it too is fraught.

One thing for sure is that, if you want to record
the same signal from a fixed pattern of dye by
integrating the output from say 10 short pulses,
as you would have recorded from a single, longer
pulse, the total number of photons in your 10
short pulses had better be the same as are
present in the long pulse. If the duty cycle
(on-time/total time) is 0.1, then the pulses must
be 10 brighter. If the duty cycle is 0.001 (10ns
on, 10 µs off) the peak brightness will be 1000x
higher. Clearly, this will bring you closer to
singlet saturation but this is only a problem in
scanned imaging as in WF, you are 5 orders of
magnitude lower in peak intensity (for the same
recorded signal, assuming 1000x1000 pixel field,
and the same frame time). So this means that the
answer to the question of "pulsed vs continuous"
may depend on WF or scanned imaging.

Now on to your suggestion: It is not obvious to
me why fewer triplets will be produced just
because you squeeze a fixed number of excitations
into a shorter time.  Surely, once excited, a
molecule has a fixed probability of decaying into
a triplet (although this probability may depend
strongly on its molecular structure and perhaps
on the milieu in which it finds itself). If we
are thinking about one-plus-one absorption events
(i.e. the re-excitation of an already excited
molecule) then re-excitation from the singlet
will be worse in the short-bright pulse (nearer
to saturation, more molecules in the siglet
already). Re-excitation from the triplet will
depend on how long it takes the singlet to decay
into a triplet as well as how long the triplet
lasts. If we assume that the decay to the triplet
is fast (<ns, which would make sense as otherwise
the singlet would have already decayed to
ground), then the reduction in the chance of a
second excitation during the "off" period will
not quite be offset by the increased chance of a
one-plus-one re-excitation during the much
brighter "on" pulse (when virtually none of the
triplets will have yet decayed, so that there are
more of them.). So I don't see how we come about
ahead with a pulse.

On the other hand, perhaps you are thinking in
terms of "Any excitation method that pushes a
significant fraction of the dye molecules into
the triplet thereby decreases the effective dye
concentration with the effect that either less
signal is produced or more excitation is needed."

Here I agree with you, but with some
qualifications. Comparing WF with confocal and
assuming for a moment, 1kx1k pixels, and a
similar collection efficiency (i.e. same optics)
and detector QE and noise (true for a disk
scanner with a good camera) and a thin specimen
(so that there is no "out-of-focus" signal to
confuse matters), then to collect an image of the
same S/N in the same time period, the confocal
excitation will have to be more intense
(photons/s/µm2) by a factor of the inverse of the
duty cycle. If we let our disk scanner have a
real Nipkov disk, which unlike a Petran disk, has
only one diffraction-limited hole in the field of
view at any one time, then the spot will cover a
minimum of about 12 Nyquist-sized pixels in the
focus plane and, as noted above will have to be
about 100,000x brighter.

We still have a choice of scan rate.

At 2 s/frame (and no scan flyback time), each
molecules will be "hit" by the "Nyquist" beam on
at least 4 different lines, during pulsed of 4,
8, 8 and 4 µs duration and about 2 ms apart (to
record the rough shape of the Airy disk: 2 pixels
in Line One, 4 in Line Two etc.). With 2ms/line,
there will be time for most triplets to decay
between pulses. On the other hand, 4 µs is long
compared to the singlet decay time (say 2ns) and
we may expect that each molecule may be excited
many times: say 100x for a total of 200ns in the
singlet-excited state during a 2-pixel, 4µs
exposure (at 5%, this is probably a bit too close
to saturation but...). Assuming that the triplet
lifetime is 10µs, then if 1% of these 100
excitations cross to the triplet, on average each
molecule will have about a 50% chance of being in
the triplet state at any given time during a
2-pixel exposure and somewhat more for 4 pixel
pulses.  Averaged over the 2µs pixel time, each
molecule will only be "available" for 2,000ns
pixel time - 200ns in singlet - 1000ns = 800 ns,
or about 40% of the time. This is a big loss.

If we scan at video rate and the pixel time drops
to about 100 ns (about 40x less than above) and
the minimum (line-scan) time between pulses drops
to about 60 µs. If we keep the laser power the
same but integrate the signal from 2s of video,
we should expose the each dye molecule to about
the same amount of excitations (neglecting the
fact that real video scan systems have low duty
cycles etc...) but, in our example, any given 2-
or 4-pixel pulse will excite each molecule 20-40x
fewer times and the chance that the effective dye
concentration will be reduced by crossing to into
the triplet during any excitation pulse will be
much lower. Maybe about 1%, i.e., not
significant. (This is one of the rationales given
for the claim that, all things being equal,
video-rate scanning produces less damage for the
same data. But "all things" are seldom equal.)

In WF, as the 100 excitations/molecule are spread
over an exposure of 2 seconds, the chance of that
any molecule will be in a triplet state at any
given time will be 1000µs/2s or essentially zero.
.
Clearly, in this example, less signal will be
lost to triplet crossing in the WF setup, but the
magnitude of the difference depends a lot on the
structure of the dye, as this determines it
probability of crossing to the triplet state and
its lifetime once there. In any case it is hard
to see how pulsing WF excitation (while making
each pulse proportionally brighter) would reduce
this problem.

The whole matter of the effect of power level,
pulse length and duty cycle on the activities of
ROS is "another problem for another hour". All we
can say here is that is likely dependent on
diffusion rates, ROS lifetimes and oxygen levels
in the cell, and I am not sure that we know
enough about these to do more than "predict what
we can measure."

Cheers,

Jim P.
*********************************************************************************
Prof. James B. Pawley,                          Ph.  608-263-3147
Room 223, Zoology Research
Building,                          FAX
608-265-5315
1117 Johnson Ave., Madison, WI, 53706  
[hidden email]
3D Microscopy of Living Cells Course, June 11-23, 2011, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/ 
Applications still being accepted
               "If it ain't diffraction, it must be statistics." Anon.

>
>
>Hi Jeremy and David,
>
>On Dec 1, 2010, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:
>
>>  On Nov 30, 2010, at 10:26 AM, Jeremy Adler wrote:
>>
>>>  *****
>>>  To join, leave or search the confocal microscopy listserv, go to:
>>>  http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>>  *****
>>>  =20
>>>  It might also be useful to have pulsatile LEDs operating in the =20
>>>  nanosecond range - there was a Stefan Hell paper that showed very =20
>>>  significant reductions in photobleaching when the gap between pulses =20=
>
>I am very interested in this, applied to wide field illumination.
>
>  >
>>  Date:    Tue, 30 Nov 2010 11:19:40 -0500
>>  From:    David Knecht <[hidden email]>
>>  Subject: Re: Guidance wanted on illumination stability
>>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>>  *****
>>
>>  It is not only possible, but Thorlabs sells an LED controller that =
>>  modulates LED intensity in this way up to 10kHz which I think should be =
>>  adequate for the effect discussed in the Hell paper.  I have not had a =
>>  chance to try it yet and see if it works to reduce bleaching.  Dave
>>  http://www.thorlabs.com/NewGroupPage9.cfm?ObjectGroup_ID=3D4003
>
>that link doesnt seem to work....
>
>or is it this?
>
>http://www.thorlabs.de/NewGroupPage9.cfm?ObjectGroup_ID=3459
>
>in any case, I'm not sure it s quite the right
>thing to avoid the bleaching and photoxicity:
>
>What we need is 10 ns ish pulses separated by
>period of zero illumination separated by 10s of
>microseconds...
>
>for example
>http://www.rapp-opto.com/index.php?show=products&id=38
>
>.. over simplified explanation below
>
>Considering the photophysics of say GFP...
>
>1) the fluorescence lifetime is a few ns,
>so intense pulses in the low ns range duration
>could put nearly all the GFPs into excited
>fluorescent state....
>but with not too much double excitation (which
>might be a bad thing - bleaching etc. )
>
>2) Next the GFP excited state relaxes in different possible ways
> a) fluorescence emission (what we want) after a a few ns
> b) non radiative energy loss (dark)
> c) inter system crossing to the triplet state
> This is bad news since:
> i) it is effectively dark
>(in the fluo emission waveband)
> ii) it is very long lived
>compared to the single excited state... T1
>lifetime is in the region of 10  micro seconds.
>
>This means that, in the typical widefield
>continuous wave illumination, especially at very
>very high illumination power,
>the triplet state is rapidly populated and
>builds up up in concentration over the exposure
>time of 10 to 100 to 1000s of milliseconds of a
>CCD camera.
>
>Potentially, much of the dye could be "trapped"
>in a dark triplet state after the first few
>milliseconds of exposure.
>And there can be multiple excitation into higher
>excited states... also bad news for
>phototoxicity and bleaching.
>
>Some (including me) seem to think that this is
>why you get more signal than you think you
>should on a fast scanning point scanner or
>spinning disk
>which give each point in the sample effectively pulsed illumination,
>and also that its the the way to prevent bleaching and phototoxicity:
>
>I mean one could use:
>1) excitation pulses that are about the same
>duration as the fluorescence lifetime (or should
>it be the fluorescence excitation time... but
>thats very short... depends on the power)
>2|) separation between the pulses of no
>excitation light power which are a bit longer
>than the triplet state lifetime...
>giving it chance to relax before it gets hit
>with another excitation photon that would bleach
>it and cause production of reactive oxygen
>species and other nastiness.
>
>So a square wave shape is needed with very long
>gaps compared to the very short pulses. ....
>and the modulated LED doesn't do that really... its a sine wave right?
>Or am I totally wrong?
>
>cheers
>
>Dan
>
>
>
>Dr. Daniel James White BSc. (Hons.) PhD
>Senior Microscopist / Image Visualisation, Processing and Analysis
>Light Microscopy and Image Processing Facilities
>Max Planck Institute of Molecular Cell Biology and Genetics
>Pfotenhauerstrasse 108
>01307 DRESDEN
>Germany
>
>[phone] (German Mobile)
>[phone] (Work phone at MPI-CBG)
>[phone] (Fax MPI-CBG LMF)
>
>http://www.bioimagexd.net        BioImageXD
>http://pacific.mpi-cbg.de                Fiji -
>is just ImageJ (Batteries Included)
>http://www.chalkie.org.uk                Dan's Homepages
>https://ifn.mpi-cbg.de                        Dresden Imaging Facility Network
>dan (at) chalkie.org.uk
>( white (at) mpi-cbg.de )

--
James and Christine Pawley, 21 N. Prospect Ave.
Madison, WI, 53726   Phone: [phone]

[Daniel James White]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Jim,

Its always a great pleasure to read your very illuminating comments (no pun intended!),

On Dec 2, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:

>
> Date:    Wed, 1 Dec 2010 13:29:40 -0600
> From:    James Pawley <[hidden email]>
> Subject: Re: Pulsed LED light sources for less photobleaching - was: Guidance wanted on illumination stability
>
> Dear Dan
>
> This matter of "pulsed vs. continuous" excitation=20
> seems to be fraught with many uncertainties.

In fact I would go further and posit that pretty much everything here is an unknown...
there are just so many variables....

Thats gonna make it hard to do systematic studies of the details...
I guess what I am getting at is just trying
pulsed wide filed illumination as some reasonable seeming pulse power, duration and separation,
and just seeing if there is a practical benefit for either or bleaching / photo toxicity.

Since A pilsed LED light source is realtively simple and inexpensive,
I think its worth a try after reading
S. Hells pulsed illumination paper from a few years ago
and  also
J Fluoresc (2009) 19:931–937 A Systematic Study on Fluorescence Enhancement under Single-photon Pulsed Illumination
Arijit Kumar De & Debabrata Goswami

both of these are from pulsed lasers...

I think that attempting to calculate the physics of this complex system
is gonna be very hard, although a greatly applaud your careful and detailed description below.

Still, since its simple, I think its work spending a little time and effort to investigate if there is an effect,
and if there is , thats great, and we can discuss how it might work after the fact.

If it does nothing - well, nothing ventured, nothing gained....

Or do you think its a waste of time, and simply can not work?

>
> How do we insure that we get the same number of=20
> excitation photons striking a fixed area of the=20
> specimen in the two modes? Do we just use a CCD=20
> camera set up to record for a fixed time period,=20
> adjust the continuous excitation level so that it=20
> produces the same signal level as we get from a=20
> pulsed illumination source, and then compare the=20
> amount of bleaching produced in each case? How=20
> are we sure that the excitation spectra are the=20
> same? We know the spectrum of a laser pretty well=20
> but an LED spectrum multiplied by the effects of=20
> an excitation filter and beamsplitter may not=20
> look the same as that which gets through these=20
> same filters from an Hg arc (which is very=20
> "spikey" in the visible). Much of the work on=20
> pulsed illumination so far has been done with=20
> 2-photon lasers which introduce a whole raft of=20
> additional unknowns (shape of pulse in time and=20
> space, 2-photon cross-section, 2-photon damage=20
> mechanisms...)

yes there is much to think about there....
but ... if once can get images of reasonable SN
compared to normal WF images, and get less bleaching/ or toxicity...
then maybe description the photo physical description can wait... interesting as it certainly would be...


Pragmatically speaking, the controlled experiment I would do would not focus on
attempting to get the same number of photon onto the sample over the course of the imaging.

instead I would go for an empirical approach, where one searches for setting that give similar S/N images,
and similar frame rates, and see of there is a n effect of bleaching / photo toxicity....
a rather biological readout...?

Not very basic physics approach I admit,
but really it the biological benefits we are chasing here?

>
> As for the photophysics, it too is fraught.
>
> One thing for sure is that, if you want to record=20
> the same signal from a fixed pattern of dye by=20
> integrating the output from say 10 short pulses,=20
> as you would have recorded from a single, longer=20
> pulse, the total number of photons in your 10=20
> short pulses had better be the same as are=20
> present in the long pulse. If the duty cycle=20
> (on-time/total time) is 0.1, then the pulses must=20
> be 10 brighter. If the duty cycle is 0.001 (10ns=20
> on, 10 =B5s off) the peak brightness will be 1000x=20
> higher. Clearly, this will bring you closer to=20
> singlet saturation but this is only a problem in=20
> scanned imaging as in WF, you are 5 orders of=20
> magnitude lower in peak intensity (for the same=20
> recorded signal, assuming 1000x1000 pixel field,=20
> and the same frame time). So this means that the=20
> answer to the question of "pulsed vs continuous"=20
> may depend on WF or scanned imaging.

Probably gonna be hard to get enough intensity from a pulsed LED to approach single saturation...
might need a flash lamp or laser (of course ) for that....
might not be impossible though.

>
> Now on to your suggestion: It is not obvious to=20
> me why fewer triplets will be produced just=20
> because you squeeze a fixed number of excitations=20
> into a shorter time.  Surely, once excited, a=20
> molecule has a fixed probability of decaying into=20
> a triplet (although this probability may depend=20
> strongly on its molecular structure and perhaps=20
> on the milieu in which it finds itself). If we=20
> are thinking about one-plus-one absorption events=20
> (i.e. the re-excitation of an already excited=20
> molecule) then re-excitation from the singlet=20
> will be worse in the short-bright pulse (nearer=20
> to saturation, more molecules in the siglet=20
> already). Re-excitation from the triplet will=20
> depend on how long it takes the singlet to decay=20
> into a triplet as well as how long the triplet=20
> lasts. If we assume that the decay to the triplet=20
> is fast (<ns, which would make sense as otherwise=20
> the singlet would have already decayed to=20
> ground), then the reduction in the chance of a=20
> second excitation during the "off" period will=20
> not quite be offset by the increased chance of a=20
> one-plus-one re-excitation during the much=20
> brighter "on" pulse (when virtually none of the=20
> triplets will have yet decayed, so that there are=20
> more of them.). So I don't see how we come about=20
> ahead with a pulse.

I did some simulations after thee ideas here:
Applied Physic Letters 92, 013902 (2008)
P.P. Mondal.
Minimizing photobleaching in fluorescence microscopy by depleting
triplet states.

There are some weaknesses in that paper, concerning not mentioning some important details...

(my python script  for the simulation is included in plain text at the bottom of this message)

... but, it seems that with CW illumination and certain combinations of lifetimes
it can be possible to deplete the ground and singlet states
and have most of the dye in the triplet (dark) state.....

of course, I am no mathematician nor physicist,
just a lowly biochemist...
so i could we way off the mark here.

I'd be very happy to be corrected.

>
> On the other hand, perhaps you are thinking in=20
> terms of "Any excitation method that pushes a=20
> significant fraction of the dye molecules into=20
> the triplet thereby decreases the effective dye=20
> concentration with the effect that either less=20
> signal is produced or more excitation is needed."

umm., in a nut shell, that exactly what I am chasing....

... plus an effect on bleaching and toxicity as a happy byproduct of that:

Less light needed to get same S/N

Right? Or do you think its doomed to failure?

cheers

Dan

>
> Here I agree with you, but with some=20
> qualifications. Comparing WF with confocal and=20
> assuming for a moment, 1kx1k pixels, and a=20
> similar collection efficiency (i.e. same optics)=20
> and detector QE and noise (true for a disk=20
> scanner with a good camera) and a thin specimen=20
> (so that there is no "out-of-focus" signal to=20
> confuse matters), then to collect an image of the=20
> same S/N in the same time period, the confocal=20
> excitation will have to be more intense=20
> (photons/s/=B5m2) by a factor of the inverse of the=20
> duty cycle. If we let our disk scanner have a=20
> real Nipkov disk, which unlike a Petran disk, has=20
> only one diffraction-limited hole in the field of=20
> view at any one time, then the spot will cover a=20
> minimum of about 12 Nyquist-sized pixels in the=20
> focus plane and, as noted above will have to be=20
> about 100,000x brighter.
>
> We still have a choice of scan rate.
>
> At 2 s/frame (and no scan flyback time), each=20
> molecules will be "hit" by the "Nyquist" beam on=20
> at least 4 different lines, during pulsed of 4,=20
> 8, 8 and 4 =B5s duration and about 2 ms apart (to=20
> record the rough shape of the Airy disk: 2 pixels=20
> in Line One, 4 in Line Two etc.). With 2ms/line,=20
> there will be time for most triplets to decay=20
> between pulses. On the other hand, 4 =B5s is long=20
> compared to the singlet decay time (say 2ns) and=20
> we may expect that each molecule may be excited=20
> many times: say 100x for a total of 200ns in the=20
> singlet-excited state during a 2-pixel, 4=B5s=20
> exposure (at 5%, this is probably a bit too close=20
> to saturation but...). Assuming that the triplet=20
> lifetime is 10=B5s, then if 1% of these 100=20
> excitations cross to the triplet, on average each=20
> molecule will have about a 50% chance of being in=20
> the triplet state at any given time during a=20
> 2-pixel exposure and somewhat more for 4 pixel=20
> pulses.  Averaged over the 2=B5s pixel time, each=20
> molecule will only be "available" for 2,000ns=20
> pixel time - 200ns in singlet - 1000ns =3D 800 ns,=20
> or about 40% of the time. This is a big loss.
>
> If we scan at video rate and the pixel time drops=20
> to about 100 ns (about 40x less than above) and=20
> the minimum (line-scan) time between pulses drops=20
> to about 60 =B5s. If we keep the laser power the=20
> same but integrate the signal from 2s of video,=20
> we should expose the each dye molecule to about=20
> the same amount of excitations (neglecting the=20
> fact that real video scan systems have low duty=20
> cycles etc...) but, in our example, any given 2-=20
> or 4-pixel pulse will excite each molecule 20-40x=20
> fewer times and the chance that the effective dye=20
> concentration will be reduced by crossing to into=20
> the triplet during any excitation pulse will be=20
> much lower. Maybe about 1%, i.e., not=20
> significant. (This is one of the rationales given=20
> for the claim that, all things being equal,=20
> video-rate scanning produces less damage for the=20
> same data. But "all things" are seldom equal.)
>
> In WF, as the 100 excitations/molecule are spread=20
> over an exposure of 2 seconds, the chance of that=20
> any molecule will be in a triplet state at any=20
> given time will be 1000=B5s/2s or essentially zero.
> =2E
> Clearly, in this example, less signal will be=20
> lost to triplet crossing in the WF setup, but the=20
> magnitude of the difference depends a lot on the=20
> structure of the dye, as this determines it=20
> probability of crossing to the triplet state and=20
> its lifetime once there. In any case it is hard=20
> to see how pulsing WF excitation (while making=20
> each pulse proportionally brighter) would reduce=20
> this problem.
>
> The whole matter of the effect of power level,=20
> pulse length and duty cycle on the activities of=20
> ROS is "another problem for another hour". All we=20
> can say here is that is likely dependent on=20
> diffusion rates, ROS lifetimes and oxygen levels=20
> in the cell, and I am not sure that we know=20
> enough about these to do more than "predict what=20
> we can measure."
>
> Cheers,
>
> Jim P.

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )





################ code begins ###########

# This code was written by Dan White at the MPI-CBG, Dresden Germany in 2010
# The license is the GPL v3

#!/usr/bin/env python
from math import *

# state equilibrium kinetic model
# s0 <=> s1   k1->   <-k2
# s1 -> t1    k3
# t1 -> s0    k4
## t1 -> bleached,  ummm lets ignore that for now.

# The steady state solution
# of the photobleaching process, according to
# Mondal, APPLIED PHYSICS LETTERS 92, 013902 (2008)
# is
# S0(t) = k4(k2 + k3) / C1
# S1(t) = fexcAk4 / C1
# and
# T1(t) = fexcAk3 / C1
# where
# C1 = fexcA(k3 + k4) + k4(k2 + k3)

# ok, so lets say fexcA = k1, for fuck's sake.


# the rate constants, per nano second

# excitation rate depends on illumnation power and absorbtion cross section of dye
# From Pawley confocal handbook pg 338 ,
# 1 mW of 488 nm laser in diffraction limited spot 10^24 photons per square cm per s
# absorption cross section of fluorecein is 3x10^-16 square cm per molecule
# so about 4x10^8 per second = 0.4 per nano second
k1 = 0.4

# fluo lifetime of fluorescein is about 5 ns, so rate is 0.2 per ns
k2 = 0.2

# ISC is also fast i guess if it happens?
# but only happens with a Quantum yeild, Qisc =  0.03... so 0.2*0.03=
k3 = 0.006

# phosphorescence and stimulated phosph rate
# phosph only is slow.... exp with O2 present , wth life time
# for GFP of 1 (to 10) micro sec (Mondal)
# for fluorescein 100 ns   (Mondal)
# so rate is 0.001 per nano sec for GFP
# this will be faster with stimulated emission, up to 0.4 or so according to stim power?
#k4 = 0.001

# pawley says steady state should be achieved (with no stim phospho)
# in about 200 ns with these rates.
# time constant to reach steady state = 1 / [t1 lifetime^-1 + (Qisc k1 k2 / (k2+k1) ) ]



# the main for loop for calc of steady state proportions of states s0, s1 and t1

for power in range (-20, 20):
    k4 = 2 ** power

    # here are the steady state equations...
   
    #C1 is the denominator
    C1 = ( k1 * (k3 + k4) ) + ( k4 *(k2 + k3) )
   
    # S0(t) = k4(k2 + k3) / C1
    s0 = ( k4 * (k2+k3) ) / C1
   
    # S1(t) = fexcAk4 / C1
    s1 = k1 * k4 / C1
   
    # T1(t) = fexcAk3 / C1
    t1 = k1 * k3 / C1
   
    print k4, s0, s1, t1, s0+s1+t1

[geekay b]

Not a confocal-related question, but I will appreciate if an
*****
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*****

Good day:

Not a confocal-related question, but I will appreciate if anyone has experience
of using Widefield Hyperspectral Imaging Systems for separating GFP and YFP
signals or autofluorescence in biological samples.


Thank you.

Sincerely
GK




________________________________
,

[Craig Brideau]

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We've done it with a laser scanning system (Nikon C1Si) but not widefield.

Craig


On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:

> Not a confocal-related question, but I will appreciate if an
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good day:
>
> Not a confocal-related question, but I will appreciate if anyone has
> experience
> of using Widefield Hyperspectral Imaging Systems for separating GFP and YFP
> signals or autofluorescence in biological samples.
>
>
> Thank you.
>
> Sincerely
> GK
>
>
>
>
> ________________________________
> ,
>
>
>

[Paul Rigby-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We've also done it in real time with the Nikon A1Si, but never widefield.

Paul Rigby

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Friday, 3 December 2010 2:58 AM
To: [hidden email]
Subject: Re: Hyperspectral imaging systems for GFP YFP

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

We've done it with a laser scanning system (Nikon C1Si) but not widefield.

Craig


On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:

> Not a confocal-related question, but I will appreciate if an
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good day:
>
> Not a confocal-related question, but I will appreciate if anyone has
> experience
> of using Widefield Hyperspectral Imaging Systems for separating GFP and YFP
> signals or autofluorescence in biological samples.
>
>
> Thank you.
>
> Sincerely
> GK
>
>
>
>
> ________________________________
> ,
>
>
>

[Daniel Gitler]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Again, Nikon C1Si. Seems to be a prevalent answer.
Daniel

----- Original Message -----
From: Paul Rigby <[hidden email]>
Date: Friday, December 3, 2010 2:19
Subject: Re: Hyperspectral imaging systems for GFP YFP
To: [hidden email]

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We've also done it in real time with the Nikon A1Si, but never
> widefield.
> Paul Rigby
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Friday, 3 December 2010 2:58 AM
> To: [hidden email]
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We've done it with a laser scanning system (Nikon C1Si) but not
> widefield.
> Craig
>
>
> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
> > Not a confocal-related question, but I will appreciate if an
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Good day:
> >
> > Not a confocal-related question, but I will appreciate if
> anyone has
> > experience
> > of using Widefield Hyperspectral Imaging Systems for
> separating GFP and YFP
> > signals or autofluorescence in biological samples.
> >
> >
> > Thank you.
> >
> > Sincerely
> > GK
> >
> >
> >
> >
> > ________________________________
> > ,
> >
> >
> >
>

Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel

Tel:  [phone]
Cell: [phone]
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎

[Andreas Bruckbauer]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****


 Would it be possible to have a widefield hyperspectral microscope? For fast aquisition you would have to split the fluorescence somhow into different channels and this is usually limited to a low number of spectral channels, like four. In principle you could couple a spectrometer to the microscope and measure one wavelength at a time but this would be slow. The confocal option seems to be more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral confocal which would be a good compromise. D. Lidke has used this system to separate 5 different quantum dots with a time resolution of 4s/frame (Immunity (2009) 31 469). The only system I know  which is widefield would be the imagestream imaging flow cytometer https://www.amnis.com/ which offers 12 simultaenous channels but in this case it is the cells which are "scanned" or flowing through the image instead of scanning the laser. Does anyone know of other options?

best wishes

Andreas


 

 

-----Original Message-----
From: Daniel Gitler <[hidden email]>
To: [hidden email]
Sent: Fri, 3 Dec 2010 6:28
Subject: Re: Hyperspectral imaging systems for GFP YFP


*****

To join, leave or search the confocal microscopy listserv, go to:

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*****



Again, Nikon C1Si. Seems to be a prevalent answer.

Daniel



----- Original Message -----

From: Paul Rigby <[hidden email]>

Date: Friday, December 3, 2010 2:19

Subject: Re: Hyperspectral imaging systems for GFP YFP

To: [hidden email]



> *****

> To join, leave or search the confocal microscopy listserv, go to:

> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

> *****

>

> We've also done it in real time with the Nikon A1Si, but never

> widefield.

> Paul Rigby

>

> -----Original Message-----

> From: Confocal Microscopy List

> [mailto:[hidden email]] On Behalf Of Craig Brideau

> Sent: Friday, 3 December 2010 2:58 AM

> To: [hidden email]

> Subject: Re: Hyperspectral imaging systems for GFP YFP

>

> *****

> To join, leave or search the confocal microscopy listserv, go to:

> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

> *****

>

> We've done it with a laser scanning system (Nikon C1Si) but not

> widefield.

> Craig

>

>

> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:

>

> > Not a confocal-related question, but I will appreciate if an

> > *****

> > To join, leave or search the confocal microscopy listserv, go to:

> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy

> > *****

> >

> > Good day:

> >

> > Not a confocal-related question, but I will appreciate if

> anyone has

> > experience

> > of using Widefield Hyperspectral Imaging Systems for

> separating GFP and YFP

> > signals or autofluorescence in biological samples.

> >

> >

> > Thank you.

> >

> > Sincerely

> > GK

> >

> >

> >

> >

> > ________________________________

> > ,

> >

> >

> >

>



Daniel Gitler, Ph.D.

Department of Physiology and Neurobiology

Faculty of Health Sciences

Ben Gurion University of the Negev

Beer-Sheva 84105

Israel



Tel:  [phone]

Cell: [phone]

Fax: + 972-8-6477628

http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎


 

[Michael Weber-4]

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Dear all,

from Robert Zucker's papers about confocal performance I recall the "Pariss Hyperspectral Imaging" system. Essentially a wide-field system equipped with a prism and a detection CCD, which allows to "slit-scan" the field of view and obtain a spectral image. No idea if this is still available, or how it performs apart from the data you can find in Robert's papers.

http://www.pariss-hyperspectral-imaging.com/

No commercial interest.

Michael


On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Would it be possible to have a widefield hyperspectral microscope? For fast aquisition you would have to split the fluorescence somhow into different channels and this is usually limited to a low number of spectral channels, like four. In principle you could couple a spectrometer to the microscope and measure one wavelength at a time but this would be slow. The confocal option seems to be more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral confocal which would be a good compromise. D. Lidke has used this system to separate 5 different quantum dots with a time resolution of 4s/frame (Immunity (2009) 31 469). The only system I know  which is widefield would be the imagestream imaging flow cytometer https://www.amnis.com/ which offers 12 simultaenous channels but in this case it is the cells which are "scanned" or flowing through the image instead of scanning the laser. Does anyone know of other options?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
> -----Original Message-----
> From: Daniel Gitler <[hidden email]>
> To: [hidden email]
> Sent: Fri, 3 Dec 2010 6:28
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Again, Nikon C1Si. Seems to be a prevalent answer.
>
> Daniel
>
>
>
> ----- Original Message -----
>
> From: Paul Rigby <[hidden email]>
>
> Date: Friday, December 3, 2010 2:19
>
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> To: [hidden email]
>
>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've also done it in real time with the Nikon A1Si, but never
>
>> widefield.
>
>> Paul Rigby
>
>>
>
>> -----Original Message-----
>
>> From: Confocal Microscopy List
>
>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>
>> Sent: Friday, 3 December 2010 2:58 AM
>
>> To: [hidden email]
>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've done it with a laser scanning system (Nikon C1Si) but not
>
>> widefield.
>
>> Craig
>
>>
>
>>
>
>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
>>
>
>>> Not a confocal-related question, but I will appreciate if an
>
>>> *****
>
>>> To join, leave or search the confocal microscopy listserv, go to:
>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>> *****
>
>>>
>
>>> Good day:
>
>>>
>
>>> Not a confocal-related question, but I will appreciate if
>
>> anyone has
>
>>> experience
>
>>> of using Widefield Hyperspectral Imaging Systems for
>
>> separating GFP and YFP
>
>>> signals or autofluorescence in biological samples.
>
>>>
>
>>>
>
>>> Thank you.
>
>>>
>
>>> Sincerely
>
>>> GK
>
>>>
>
>>>
>
>>>
>
>>>
>
>>> ________________________________
>
>>> ,
>
>>>
>
>>>
>
>>>
>
>>
>
>
>
> Daniel Gitler, Ph.D.
>
> Department of Physiology and Neurobiology
>
> Faculty of Health Sciences
>
> Ben Gurion University of the Negev
>
> Beer-Sheva 84105
>
> Israel
>
>
>
> Tel:  [phone]
>
> Cell: [phone]
>
> Fax: + 972-8-6477628
>
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>

[geekay b]

Thanks to all the replies and suggestions.

I am more in
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Good day:

Thanks to all the replies and suggestions.

I am more interested in knowing the abilities of widefield (low cost)
hyperspectral imaging systems, such as the Pariss one mentioned by Michael
or other such systems in resolving such signals. I think there are a few
commercial systems out there?

Thanks again,
GK



________________________________
From: Michael Weber <[hidden email]>
To: [hidden email]
Sent: Fri, December 3, 2010 5:29:49 AM
Subject: Re: Hyperspectral imaging systems for GFP YFP

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear all,

from Robert Zucker's papers about confocal performance I recall the "Pariss
Hyperspectral Imaging" system. Essentially a wide-field system equipped with a
prism and a detection CCD, which allows to "slit-scan" the field of view and
obtain a spectral image. No idea if this is still available, or how it performs
apart from the data you can find in Robert's papers.

http://www.pariss-hyperspectral-imaging.com/

No commercial interest.

Michael


On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Would it be possible to have a widefield hyperspectral microscope? For fast
>aquisition you would have to split the fluorescence somhow into different
>channels and this is usually limited to a low number of spectral channels, like
>four. In principle you could couple a spectrometer to the microscope and measure
>one wavelength at a time but this would be slow. The confocal option seems to be
>more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et
>al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral
>confocal which would be a good compromise. D. Lidke has used this system to
>separate 5 different quantum dots with a time resolution of 4s/frame (Immunity
>(2009) 31 469). The only system I know  which is widefield would be the
>imagestream imaging flow cytometer https://www.amnis.com/ which offers 12
>simultaenous channels but in this case it is the cells which are "scanned" or
>flowing through the image instead of scanning the laser. Does anyone know of
>other options?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
> -----Original Message-----
> From: Daniel Gitler <[hidden email]>
> To: [hidden email]
> Sent: Fri, 3 Dec 2010 6:28
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Again, Nikon C1Si. Seems to be a prevalent answer.
>
> Daniel
>
>
>
> ----- Original Message -----
>
> From: Paul Rigby <[hidden email]>
>
> Date: Friday, December 3, 2010 2:19
>
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> To: [hidden email]
>
>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've also done it in real time with the Nikon A1Si, but never
>
>> widefield.
>
>> Paul Rigby
>
>>
>
>> -----Original Message-----
>
>> From: Confocal Microscopy List
>
>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>
>> Sent: Friday, 3 December 2010 2:58 AM
>
>> To: [hidden email]
>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've done it with a laser scanning system (Nikon C1Si) but not
>
>> widefield.
>
>> Craig
>
>>
>
>>
>
>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
>>
>
>>> Not a confocal-related question, but I will appreciate if an
>
>>> *****
>
>>> To join, leave or search the confocal microscopy listserv, go to:
>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>> *****
>
>>>
>
>>> Good day:
>
>>>
>
>>> Not a confocal-related question, but I will appreciate if
>
>> anyone has
>
>>> experience
>
>>> of using Widefield Hyperspectral Imaging Systems for
>
>> separating GFP and YFP
>
>>> signals or autofluorescence in biological samples.
>
>>>
>
>>>
>
>>> Thank you.
>
>>>
>
>>> Sincerely
>
>>> GK
>
>>>
>
>>>
>
>>>
>
>>>
>
>>> ________________________________
>
>>> ,
>
>>>
>
>>>
>
>>>
>
>>
>
>
>
> Daniel Gitler, Ph.D.
>
> Department of Physiology and Neurobiology
>
> Faculty of Health Sciences
>
> Ben Gurion University of the Negev
>
> Beer-Sheva 84105
>
> Israel
>
>
>
> Tel:  [phone]
>
> Cell: [phone]
>
> Fax: + 972-8-6477628
>
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>

[Julio Vazquez]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

You may also check CRI:

http://www.cri-inc.com/

They offer a couple of "multispectral" or "hyperspectral" imaging  
devices that can be mounted on conventional microscopes.

-No commercial interest-

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

http://www.fhcrc.org



On Dec 3, 2010, at 5:29 AM, Michael Weber wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> from Robert Zucker's papers about confocal performance I recall the  
> "Pariss Hyperspectral Imaging" system. Essentially a wide-field  
> system equipped with a prism and a detection CCD, which allows to  
> "slit-scan" the field of view and obtain a spectral image. No idea  
> if this is still available, or how it performs apart from the data  
> you can find in Robert's papers.
>
> http://www.pariss-hyperspectral-imaging.com/
>
> No commercial interest.
>
> Michael
>
>
> On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> Would it be possible to have a widefield hyperspectral microscope?  
>> For fast aquisition you would have to split the fluorescence  
>> somhow into different channels and this is usually limited to a  
>> low number of spectral channels, like four. In principle you could  
>> couple a spectrometer to the microscope and measure one wavelength  
>> at a time but this would be slow. The confocal option seems to be  
>> more apealing, is the Nikon C1si a point or slit scanning system?  
>> M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit  
>> scanning hyperspectral confocal which would be a good compromise.  
>> D. Lidke has used this system to separate 5 different quantum dots  
>> with a time resolution of 4s/frame (Immunity (2009) 31 469). The  
>> only system I know  which is widefield would be the imagestream  
>> imaging flow cytometer https://www.amnis.com/ which offers 12  
>> simultaenous channels but in this case it is the cells which are  
>> "scanned" or flowing through the image instead of scanning the  
>> laser. Does anyone know of other options?
>>
>> best wishes
>>
>> Andreas
>>
>>
>>
>>
>>
>>
>> -----Original Message-----
>> From: Daniel Gitler <[hidden email]>
>> To: [hidden email]
>> Sent: Fri, 3 Dec 2010 6:28
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>>
>>
>> *****
>>
>> To join, leave or search the confocal microscopy listserv, go to:
>>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>
>> *****
>>
>>
>>
>> Again, Nikon C1Si. Seems to be a prevalent answer.
>>
>> Daniel
>>
>>
>>
>> ----- Original Message -----
>>
>> From: Paul Rigby <[hidden email]>
>>
>> Date: Friday, December 3, 2010 2:19
>>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>>
>> To: [hidden email]
>>
>>
>>
>>> *****
>>
>>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>
>>> *****
>>
>>>
>>
>>> We've also done it in real time with the Nikon A1Si, but never
>>
>>> widefield.
>>
>>> Paul Rigby
>>
>>>
>>
>>> -----Original Message-----
>>
>>> From: Confocal Microscopy List
>>
>>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>>
>>> Sent: Friday, 3 December 2010 2:58 AM
>>
>>> To: [hidden email]
>>
>>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>>
>>>
>>
>>> *****
>>
>>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>
>>> *****
>>
>>>
>>
>>> We've done it with a laser scanning system (Nikon C1Si) but not
>>
>>> widefield.
>>
>>> Craig
>>
>>>
>>
>>>
>>
>>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>>
>>>
>>
>>>> Not a confocal-related question, but I will appreciate if an
>>
>>>> *****
>>
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>
>>>> *****
>>
>>>>
>>
>>>> Good day:
>>
>>>>
>>
>>>> Not a confocal-related question, but I will appreciate if
>>
>>> anyone has
>>
>>>> experience
>>
>>>> of using Widefield Hyperspectral Imaging Systems for
>>
>>> separating GFP and YFP
>>
>>>> signals or autofluorescence in biological samples.
>>
>>>>
>>
>>>>
>>
>>>> Thank you.
>>
>>>>
>>
>>>> Sincerely
>>
>>>> GK
>>
>>>>
>>
>>>>
>>
>>>>
>>
>>>>
>>
>>>> ________________________________
>>
>>>> ,
>>
>>>>
>>
>>>>
>>
>>>>
>>
>>>
>>
>>
>>
>> Daniel Gitler, Ph.D.
>>
>> Department of Physiology and Neurobiology
>>
>> Faculty of Health Sciences
>>
>> Ben Gurion University of the Negev
>>
>> Beer-Sheva 84105
>>
>> Israel
>>
>>
>>
>> Tel:  [phone]
>>
>> Cell: [phone]
>>
>> Fax: + 972-8-6477628
>>
>> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>>

[Kurt Thorn]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

  I have some experience with this widefield hyperspectral imaging
system, which is based on a tunable AOTF:
http://www.olinet.com/products.php?&_act=manageProduct&DO=viewProduct&intProdID=53&product=Hyper/Multispectral%20Imaging%20System

It works well - continuously tunable center wavelength, multiple
different widths for each passband, and very fast retuning from one
wavelength to the next, but the out of band rejection of ~1:10^3 is
non-ideal.  Of course, you also have to be willing to acquire your
spectral images sequentially rather than simultaneously.

There are also tunable liquid crystal filters, but they take longer (100
ms?) to change wavelengths.

For the application we're interested in, we've actually decided it's
easier just to use a filter wheel and acquire hyperspectral images by
using multiple bandpass filters.

Kurt

On 12/2/2010 11:10 PM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
>   Would it be possible to have a widefield hyperspectral microscope? For fast aquisition you would have to split the fluorescence somhow into different channels and this is usually limited to a low number of spectral channels, like four. In principle you could couple a spectrometer to the microscope and measure one wavelength at a time but this would be slow. The confocal option seems to be more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral confocal which would be a good compromise. D. Lidke has used this system to separate 5 different quantum dots with a time resolution of 4s/frame (Immunity (2009) 31 469). The only system I know  which is widefield would be the imagestream imaging flow cytometer https://www.amnis.com/ which offers 12 simultaenous channels but in this case it is the cells which are "scanned" or flowing through the image instead of scanning the laser. Does anyone know of other options?
>
> best wishes
>
> Andreas

[Alex Fong-2]

[Note - Commercial Post]

Hi Daniel,

Our HSi-300/400 spectral imagers are wide field systems designed for performing unmixing of FP signals and auto-fluorescence. In fact you may want to check this article from a Mayo Clinic team discuss just that:

" Cayle S. Lisenbee, Kaleeckal G. Harikumar, Laurence J. Miller", PROBING THE ARCHITECTURE OF CHAMELEON SECRETIN RECEPTORS WITH FRET USING AOTF-BASED SPECTRAL IMAGING

http://www.goochandhousego.com/sites/default/files/documents/Probing_the_Architecture_of_Chameleon_Secretins.pdf 

More details on the system here:

http://www.goochandhousego.com/products/life-science-instrumentation/spectral-imaging-synthesis/hsi-300-hyper/multispectral-imaging 

Give me a call or email me if you have any questions!

Best regards,

Alexandre Fong
Senior Vice-President, Life Sciences and Instrumentation
Gooch & Housego (Orlando), LLC.
4632 36th Street,
Orlando, FL 32811
Ph: 407-422-3171 Ext. 202
Mobile: [phone]
Fax: [phone]
Email: [hidden email]
Web : http://www.goochandhousego.com/products/systems
Web - Alternate: http://www.olinet.com 
Skype: alexandre.fong 
Alternate Email: [hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Weber
Sent: Friday, December 03, 2010 8:30 AM
To: [hidden email]
Subject: Re: Hyperspectral imaging systems for GFP YFP

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear all,

from Robert Zucker's papers about confocal performance I recall the "Pariss Hyperspectral Imaging" system. Essentially a wide-field system equipped with a prism and a detection CCD, which allows to "slit-scan" the field of view and obtain a spectral image. No idea if this is still available, or how it performs apart from the data you can find in Robert's papers.

http://www.pariss-hyperspectral-imaging.com/

No commercial interest.

Michael


On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Would it be possible to have a widefield hyperspectral microscope? For fast aquisition you would have to split the fluorescence somhow into different channels and this is usually limited to a low number of spectral channels, like four. In principle you could couple a spectrometer to the microscope and measure one wavelength at a time but this would be slow. The confocal option seems to be more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral confocal which would be a good compromise. D. Lidke has used this system to separate 5 different quantum dots with a time resolution of 4s/frame (Immunity (2009) 31 469). The only system I know  which is widefield would be the imagestream imaging flow cytometer https://www.amnis.com/ which offers 12 simultaenous channels but in this case it is the cells which are "scanned" or flowing through the image instead of scanning the laser. Does anyone know of other options?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
> -----Original Message-----
> From: Daniel Gitler <[hidden email]>
> To: [hidden email]
> Sent: Fri, 3 Dec 2010 6:28
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Again, Nikon C1Si. Seems to be a prevalent answer.
>
> Daniel
>
>
>
> ----- Original Message -----
>
> From: Paul Rigby <[hidden email]>
>
> Date: Friday, December 3, 2010 2:19
>
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> To: [hidden email]
>
>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've also done it in real time with the Nikon A1Si, but never
>
>> widefield.
>
>> Paul Rigby
>
>>
>
>> -----Original Message-----
>
>> From: Confocal Microscopy List
>
>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>
>> Sent: Friday, 3 December 2010 2:58 AM
>
>> To: [hidden email]
>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've done it with a laser scanning system (Nikon C1Si) but not
>
>> widefield.
>
>> Craig
>
>>
>
>>
>
>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
>>
>
>>> Not a confocal-related question, but I will appreciate if an
>
>>> *****
>
>>> To join, leave or search the confocal microscopy listserv, go to:
>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>> *****
>
>>>
>
>>> Good day:
>
>>>
>
>>> Not a confocal-related question, but I will appreciate if
>
>> anyone has
>
>>> experience
>
>>> of using Widefield Hyperspectral Imaging Systems for
>
>> separating GFP and YFP
>
>>> signals or autofluorescence in biological samples.
>
>>>
>
>>>
>
>>> Thank you.
>
>>>
>
>>> Sincerely
>
>>> GK
>
>>>
>
>>>
>
>>>
>
>>>
>
>>> ________________________________
>
>>> ,
>
>>>
>
>>>
>
>>>
>
>>
>
>
>
> Daniel Gitler, Ph.D.
>
> Department of Physiology and Neurobiology
>
> Faculty of Health Sciences
>
> Ben Gurion University of the Negev
>
> Beer-Sheva 84105
>
> Israel
>
>
>
> Tel:  [phone]
>
> Cell: [phone]
>
> Fax: + 972-8-6477628
>
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>

[Alex Fong-2]

[Note - Commercial Post]
[Apologies for the last cryptic post - confirmed I am sending this one as 'Plain Text']

Hi Daniel,

Our HSi-300/400 spectral imagers are wide field systems designed for performing unmixing of FP signals and auto-fluorescence. In fact you may want to check this article from a Mayo Clinic team discuss just that:

" Cayle S. Lisenbee, Kaleeckal G. Harikumar, Laurence J. Miller", PROBING THE ARCHITECTURE OF CHAMELEON SECRETIN RECEPTORS WITH FRET USING AOTF-BASED SPECTRAL IMAGING

http://www.goochandhousego.com/sites/default/files/documents/Probing_the_Architecture_of_Chameleon_Secretins.pdf 

More details on the system here:

http://www.goochandhousego.com/products/life-science-instrumentation/spectral-imaging-synthesis/hsi-300-hyper/multispectral-imaging 

Give me a call or email me if you have any questions!

Best regards,

Alexandre Fong
Senior Vice-President, Life Sciences and Instrumentation
Gooch & Housego (Orlando), LLC.
4632 36th Street,
Orlando, FL 32811
Ph: 407-422-3171 Ext. 202
Mobile: [phone]
Fax: [phone]
Email: [hidden email]
Web : http://www.goochandhousego.com/products/systems
Web - Alternate: http://www.olinet.com 
Skype: alexandre.fong 
Alternate Email: [hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Weber
Sent: Friday, December 03, 2010 8:30 AM
To: [hidden email]
Subject: Re: Hyperspectral imaging systems for GFP YFP

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear all,

from Robert Zucker's papers about confocal performance I recall the "Pariss Hyperspectral Imaging" system. Essentially a wide-field system equipped with a prism and a detection CCD, which allows to "slit-scan" the field of view and obtain a spectral image. No idea if this is still available, or how it performs apart from the data you can find in Robert's papers.

http://www.pariss-hyperspectral-imaging.com/

No commercial interest.

Michael


On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> Would it be possible to have a widefield hyperspectral microscope? For fast aquisition you would have to split the fluorescence somhow into different channels and this is usually limited to a low number of spectral channels, like four. In principle you could couple a spectrometer to the microscope and measure one wavelength at a time but this would be slow. The confocal option seems to be more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral confocal which would be a good compromise. D. Lidke has used this system to separate 5 different quantum dots with a time resolution of 4s/frame (Immunity (2009) 31 469). The only system I know  which is widefield would be the imagestream imaging flow cytometer https://www.amnis.com/ which offers 12 simultaenous channels but in this case it is the cells which are "scanned" or flowing through the image instead of scanning the laser. Does anyone know of other options?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
> -----Original Message-----
> From: Daniel Gitler <[hidden email]>
> To: [hidden email]
> Sent: Fri, 3 Dec 2010 6:28
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> *****
>
>
>
> Again, Nikon C1Si. Seems to be a prevalent answer.
>
> Daniel
>
>
>
> ----- Original Message -----
>
> From: Paul Rigby <[hidden email]>
>
> Date: Friday, December 3, 2010 2:19
>
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> To: [hidden email]
>
>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've also done it in real time with the Nikon A1Si, but never
>
>> widefield.
>
>> Paul Rigby
>
>>
>
>> -----Original Message-----
>
>> From: Confocal Microscopy List
>
>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>
>> Sent: Friday, 3 December 2010 2:58 AM
>
>> To: [hidden email]
>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>> *****
>
>>
>
>> We've done it with a laser scanning system (Nikon C1Si) but not
>
>> widefield.
>
>> Craig
>
>>
>
>>
>
>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
>>
>
>>> Not a confocal-related question, but I will appreciate if an
>
>>> *****
>
>>> To join, leave or search the confocal microscopy listserv, go to:
>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
>>> *****
>
>>>
>
>>> Good day:
>
>>>
>
>>> Not a confocal-related question, but I will appreciate if
>
>> anyone has
>
>>> experience
>
>>> of using Widefield Hyperspectral Imaging Systems for
>
>> separating GFP and YFP
>
>>> signals or autofluorescence in biological samples.
>
>>>
>
>>>
>
>>> Thank you.
>
>>>
>
>>> Sincerely
>
>>> GK
>
>>>
>
>>>
>
>>>
>
>>>
>
>>> ________________________________
>
>>> ,
>
>>>
>
>>>
>
>>>
>
>>
>
>
>
> Daniel Gitler, Ph.D.
>
> Department of Physiology and Neurobiology
>
> Faculty of Health Sciences
>
> Ben Gurion University of the Negev
>
> Beer-Sheva 84105
>
> Israel
>
>
>
> Tel:  [phone]
>
> Cell: [phone]
>
> Fax: + 972-8-6477628
>
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
>

[Louis Kerr]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

We have experience with the Lightform PARISS system - http://www.lightforminc.com/ 

and the CRI Nuance system for widefield systems - http://www.cri-inc.com/products/nuance.asp 

We have also used the Nikon C1 and Zeiss LSM 710 confocal systems.

They all work well and have plenty of overlap in capabilities but each has particular strengths.

I guess we should write a review sometime!

Thanks,
Louie

----- Original Message -----
From: "geekay b" <[hidden email]>
To: [hidden email]
Sent: Friday, December 3, 2010 12:26:40 PM
Subject: Re: Hyperspectral imaging systems for GFP YFP

Thanks to all the replies and suggestions.

I am more interested in knowing the abilities of widefield (low cost)
hyperspectral imaging systems, such as the Pariss one mentioned by Michael
or other such systems in resolving such signals. I think there are a few
commercial systems out there?

Thanks again,
GK

________________________________
From: Michael Weber <[hidden email]>
To: [hidden email]
Sent: Fri, December 3, 2010 5:29:49 AM
Subject: Re: Hyperspectral imaging systems for GFP YFP

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
*****

Dear all,

from Robert Zucker's papers about confocal performance I recall the "Pariss
Hyperspectral Imaging" system. Essentially a wide-field system equipped with a
prism and a detection CCD, which allows to "slit-scan" the field of view and
obtain a spectral image. No idea if this is still available, or how it performs
apart from the data you can find in Robert's papers.

http://www.pariss-hyperspectral-imaging.com/ 

No commercial interest.

Michael


On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
> *****
>
>
> Would it be possible to have a widefield hyperspectral microscope? For fast
>aquisition you would have to split the fluorescence somhow into different
>channels and this is usually limited to a low number of spectral channels, like
>four. In principle you could couple a spectrometer to the microscope and measure
>one wavelength at a time but this would be slow. The confocal option seems to be
>more apealing, is the Nikon C1si a point or slit scanning system? M. Sinclair et
>al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral
>confocal which would be a good compromise. D. Lidke has used this system to
>separate 5 different quantum dots with a time resolution of 4s/frame (Immunity
>(2009) 31 469). The only system I know which is widefield would be the
>imagestream imaging flow cytometer https://www.amnis.com/ which offers 12
>simultaenous channels but in this case it is the cells which are "scanned" or
>flowing through the image instead of scanning the laser. Does anyone know of
>other options?
>
> best wishes
>
> Andreas
>
>
>
>
>
>
> -----Original Message-----
> From: Daniel Gitler <[hidden email]>
> To: [hidden email]
> Sent: Fri, 3 Dec 2010 6:28
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>
> *****
>
> To join, leave or search the confocal microscopy listserv, go to:
>
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>
> *****
>
>
>
> Again, Nikon C1Si. Seems to be a prevalent answer.
>
> Daniel
>
>
>
> ----- Original Message -----
>
> From: Paul Rigby <[hidden email]>
>
> Date: Friday, December 3, 2010 2:19
>
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> To: [hidden email]
>
>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>
>> *****
>
>>
>
>> We've also done it in real time with the Nikon A1Si, but never
>
>> widefield.
>
>> Paul Rigby
>
>>
>
>> -----Original Message-----
>
>> From: Confocal Microscopy List
>
>> [mailto:[hidden email]] On Behalf Of Craig Brideau
>
>> Sent: Friday, 3 December 2010 2:58 AM
>
>> To: [hidden email]
>
>> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
>>
>
>> *****
>
>> To join, leave or search the confocal microscopy listserv, go to:
>
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>
>> *****
>
>>
>
>> We've done it with a laser scanning system (Nikon C1Si) but not
>
>> widefield.
>
>> Craig
>
>>
>
>>
>
>> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
>
>>
>
>>> Not a confocal-related question, but I will appreciate if an
>
>>> *****
>
>>> To join, leave or search the confocal microscopy listserv, go to:
>
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy 
>
>>> *****
>
>>>
>
>>> Good day:
>
>>>
>
>>> Not a confocal-related question, but I will appreciate if
>
>> anyone has
>
>>> experience
>
>>> of using Widefield Hyperspectral Imaging Systems for
>
>> separating GFP and YFP
>
>>> signals or autofluorescence in biological samples.
>
>>>
>
>>>
>
>>> Thank you.
>
>>>
>
>>> Sincerely
>
>>> GK
>
>>>
>
>>>
>
>>>
>
>>>
>
>>> ________________________________
>
>>> ,
>
>>>
>
>>>
>
>>>
>
>>
>
>
>
> Daniel Gitler, Ph.D.
>
> Department of Physiology and Neurobiology
>
> Faculty of Health Sciences
>
> Ben Gurion University of the Negev
>
> Beer-Sheva 84105
>
> Israel
>
>
>
> Tel: [phone]
>
> Cell: [phone]
>
> Fax: + 972-8-6477628
>
> http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎ 
>

--
Louis Kerr
[hidden email]

Research and Education Support Coordinator
Marine Biological Laboratory
7 MBL Street
Woods Hole, MA 02543
508-289-7273
508-540-6902 (FAX)
508-292-0289 (Cell phone)

VISIT OUR WEB SITES:
http://www.mbl.edu/ 
http://www.courses.mbl.edu/ 

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

It sounds like you have had the chance to try out quite a variety of
systems!  It would be interesting to hear your comparison of them.

Craig


On Mon, Dec 6, 2010 at 4:05 PM, Louis Kerr <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We have experience with the Lightform PARISS system -
> http://www.lightforminc.com/
>
> and the CRI Nuance system for widefield systems -
> http://www.cri-inc.com/products/nuance.asp
>
> We have also used the Nikon C1 and Zeiss LSM 710 confocal systems.
>
> They all work well and have plenty of overlap in capabilities but each has
> particular strengths.
>
> I guess we should write a review sometime!
>
> Thanks,
> Louie
>
> ----- Original Message -----
> From: "geekay b" <[hidden email]>
> To: [hidden email]
> Sent: Friday, December 3, 2010 12:26:40 PM
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> Thanks to all the replies and suggestions.
>
> I am more interested in knowing the abilities of widefield (low cost)
> hyperspectral imaging systems, such as the Pariss one mentioned by Michael
> or other such systems in resolving such signals. I think there are a few
> commercial systems out there?
>
> Thanks again,
> GK
>
> ________________________________
> From: Michael Weber <[hidden email]>
> To: [hidden email]
> Sent: Fri, December 3, 2010 5:29:49 AM
> Subject: Re: Hyperspectral imaging systems for GFP YFP
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> from Robert Zucker's papers about confocal performance I recall the "Pariss
> Hyperspectral Imaging" system. Essentially a wide-field system equipped
> with a
> prism and a detection CCD, which allows to "slit-scan" the field of view
> and
> obtain a spectral image. No idea if this is still available, or how it
> performs
> apart from the data you can find in Robert's papers.
>
> http://www.pariss-hyperspectral-imaging.com/
>
> No commercial interest.
>
> Michael
>
>
> On Dec 3, 2010, at 8:10 AM, Andreas Bruckbauer wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> >
> > Would it be possible to have a widefield hyperspectral microscope? For
> fast
> >aquisition you would have to split the fluorescence somhow into different
> >channels and this is usually limited to a low number of spectral channels,
> like
> >four. In principle you could couple a spectrometer to the microscope and
> measure
> >one wavelength at a time but this would be slow. The confocal option seems
> to be
> >more apealing, is the Nikon C1si a point or slit scanning system? M.
> Sinclair et
> >al. Applied Optics (2006) 45 6283 have build a slit scanning hyperspectral
> >confocal which would be a good compromise. D. Lidke has used this system
> to
> >separate 5 different quantum dots with a time resolution of 4s/frame
> (Immunity
> >(2009) 31 469). The only system I know which is widefield would be the
> >imagestream imaging flow cytometer https://www.amnis.com/ which offers 12
> >simultaenous channels but in this case it is the cells which are "scanned"
> or
> >flowing through the image instead of scanning the laser. Does anyone know
> of
> >other options?
> >
> > best wishes
> >
> > Andreas
> >
> >
> >
> >
> >
> >
> > -----Original Message-----
> > From: Daniel Gitler <[hidden email]>
> > To: [hidden email]
> > Sent: Fri, 3 Dec 2010 6:28
> > Subject: Re: Hyperspectral imaging systems for GFP YFP
> >
> >
> > *****
> >
> > To join, leave or search the confocal microscopy listserv, go to:
> >
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> > *****
> >
> >
> >
> > Again, Nikon C1Si. Seems to be a prevalent answer.
> >
> > Daniel
> >
> >
> >
> > ----- Original Message -----
> >
> > From: Paul Rigby <[hidden email]>
> >
> > Date: Friday, December 3, 2010 2:19
> >
> > Subject: Re: Hyperspectral imaging systems for GFP YFP
> >
> > To: [hidden email]
> >
> >
> >
> >> *****
> >
> >> To join, leave or search the confocal microscopy listserv, go to:
> >
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> >> *****
> >
> >>
> >
> >> We've also done it in real time with the Nikon A1Si, but never
> >
> >> widefield.
> >
> >> Paul Rigby
> >
> >>
> >
> >> -----Original Message-----
> >
> >> From: Confocal Microscopy List
> >
> >> [mailto:[hidden email]] On Behalf Of Craig Brideau
> >
> >> Sent: Friday, 3 December 2010 2:58 AM
> >
> >> To: [hidden email]
> >
> >> Subject: Re: Hyperspectral imaging systems for GFP YFP
> >
> >>
> >
> >> *****
> >
> >> To join, leave or search the confocal microscopy listserv, go to:
> >
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> >> *****
> >
> >>
> >
> >> We've done it with a laser scanning system (Nikon C1Si) but not
> >
> >> widefield.
> >
> >> Craig
> >
> >>
> >
> >>
> >
> >> On Thu, Dec 2, 2010 at 11:30 AM, geekay b <[hidden email]> wrote:
> >
> >>
> >
> >>> Not a confocal-related question, but I will appreciate if an
> >
> >>> *****
> >
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >
> >>> *****
> >
> >>>
> >
> >>> Good day:
> >
> >>>
> >
> >>> Not a confocal-related question, but I will appreciate if
> >
> >> anyone has
> >
> >>> experience
> >
> >>> of using Widefield Hyperspectral Imaging Systems for
> >
> >> separating GFP and YFP
> >
> >>> signals or autofluorescence in biological samples.
> >
> >>>
> >
> >>>
> >
> >>> Thank you.
> >
> >>>
> >
> >>> Sincerely
> >
> >>> GK
> >
> >>>
> >
> >>>
> >
> >>>
> >
> >>>
> >
> >>> ________________________________
> >
> >>> ,
> >
> >>>
> >
> >>>
> >
> >>>
> >
> >>
> >
> >
> >
> > Daniel Gitler, Ph.D.
> >
> > Department of Physiology and Neurobiology
> >
> > Faculty of Health Sciences
> >
> > Ben Gurion University of the Negev
> >
> > Beer-Sheva 84105
> >
> > Israel
> >
> >
> >
> > Tel: [phone]
> >
> > Cell: [phone]
> >
> > Fax: + 972-8-6477628
> >
> > http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/‎
> >
>
>
>
>
>
> --
> Louis Kerr
> [hidden email]
>
> Research and Education Support Coordinator
> Marine Biological Laboratory
> 7 MBL Street
> Woods Hole, MA 02543
> 508-289-7273
> 508-540-6902 (FAX)
> 508-292-0289 (Cell phone)
>
> VISIT OUR WEB SITES:
> http://www.mbl.edu/
> http://www.courses.mbl.edu/
>