Spectral: Single channel vs Multi-channel

[Richard Cole]

I looked very carefully at both systems and my conclusion are:

If you need speed a single channel, a multi-channel spectral PMT is the way to go.

If speed is not an issue, as I my case, the scanning system offers a couple of major advantages

1. For spectral unmixing the dyes can have greatly different fluorescent levels (I have separated two over lapping fluorophores with 10X concentration difference)

2. The size of the slit and the number of sampling points are user adjustable.  

Just my 2c


Rich

Richard Cole
Research Scientist V
Director: Advanced Light Microscopy & Image Analysis Core
Wadsworth Center
P.O. Box 509 Albany N.Y. 12201-0509
 
Research Assistant Professor
Dept. of Biomedical Sciences
School of Public Health State University of New York Albany, New York

SS518-474-7048
 518-474-4430  
  [hidden email]
Website www.wadsworth.org/cores/alm/index.htm


________________________________________
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of CONFOCALMICROSCOPY automatic digest system
Sent: Sunday, April 12, 2009 1:05 AM
To: [hidden email]
Subject: CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)



 

CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
Table of contents:
• Spectral: Single channel vs Multi-channel
1. Spectral: Single channel vs Multi-channel
o Spectral: Single channel vs Multi-channel (04/11)
From: "Richard E. Edelmann" <[hidden email]>


Browse the CONFOCALMICROSCOPY online archives.





IMPORTANT NOTICE: This e-mail and any attachments may contain
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[Badri Roysam]

While on this topic, I am always to eager to learn about ways to image many more
channels at a time. Has anyone made any breakthroughs lately that may permit us to
stain and image, say 10 - 20 channels...?


Badri Roysam
Professor, Department of Electrical, Computer and Systems Engineering
Associate Director, NSF Center for Subsurface Sensing & Imaging Systems (CenSSIS ERC)
Co-Director, Rensselaer Center for Open Source Software
Rensselaer Polytechnic Institute
110 8th Street, Troy, New York 12180-3590, USA.
Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308): 518-276-8207, Fax: [phone]
Email: [hidden email], Web: http://www.ecse.rpi.edu/~roysam



----- Original Message -----
From: Richard Cole [mailto:[hidden email]]
To: [hidden email]
Subject: Spectral: Single channel vs Multi-channel

> I looked very carefully at both systems and my conclusion are:
>
> If you need speed a single channel, a multi-channel spectral PMT is the way
> to go.
>
> If speed is not an issue, as I my case, the scanning system offers a couple
> of major advantages
>
> 1. For spectral unmixing the dyes can have greatly different fluorescent
> levels (I have separated two over lapping fluorophores with 10X
> concentration difference)
>
> 2. The size of the slit and the number of sampling points are user
> adjustable.  
>
> Just my 2c
>
>
> Rich
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
> P.O. Box 509 Albany N.Y. 12201-0509
>  
> Research Assistant Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York Albany, New York
>
> SS518-474-7048
>  518-474-4430  
>   [hidden email]
> Website www.wadsworth.org/cores/alm/index.htm
>
>
> ________________________________________
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of CONFOCALMICROSCOPY automatic digest system
> Sent: Sunday, April 12, 2009 1:05 AM
> To: [hidden email]
> Subject: CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
>
>
>
>  
>
> CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
> Table of contents:
> • Spectral: Single channel vs Multi-channel
> 1. Spectral: Single channel vs Multi-channel
> o Spectral: Single channel vs Multi-channel (04/11)
> From: "Richard E. Edelmann" <[hidden email]>
>
>
> Browse the CONFOCALMICROSCOPY online archives.
>
>
>
>
>
> IMPORTANT NOTICE: This e-mail and any attachments may contain
> confidential or sensitive information which is, or may be, legally
> privileged or otherwise protected by law from further disclosure.  It
> is intended only for the addressee.  If you received this in error or
> from someone who was not authorized to send it to you, please do not
> distribute, copy or use it or any attachments.  Please notify the
> sender immediately by reply e-mail and delete this from your
> system. Thank you for your cooperation.
>

[Craig Brideau]

The Nikon C1Si will do that with spectral unmixing.  Speed is about ~1fps @512x512 pixels.

Craig

On Mon, Apr 13, 2009 at 6:55 AM, Badri Roysam <[hidden email]> wrote:

While on this topic, I am always to eager to learn about ways to image many more
channels at a time. Has anyone made any breakthroughs lately that may permit us to
stain and image, say 10 - 20 channels...?


Badri Roysam
Professor, Department of Electrical, Computer and Systems Engineering
Associate Director, NSF Center for Subsurface Sensing & Imaging Systems (CenSSIS ERC)
Co-Director, Rensselaer Center for Open Source Software
Rensselaer Polytechnic Institute
110 8th Street, Troy, New York 12180-3590, USA.
Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308): 518-276-8207, Fax: [phone]
Email: [hidden email], Web: http://www.ecse.rpi.edu/~roysam

----- Original Message -----
From: Richard Cole [mailto:[hidden email]]
To: [hidden email]

Subject: Spectral: Single channel vs Multi-channel


> I looked very carefully at both systems and my conclusion are:
>
> If you need speed a single channel, a multi-channel spectral PMT is the way
> to go.
>
> If speed is not an issue, as I my case, the scanning system offers a couple
> of major advantages
>
> 1. For spectral unmixing the dyes can have greatly different fluorescent
> levels (I have separated two over lapping fluorophores with 10X
> concentration difference)
>
> 2. The size of the slit and the number of sampling points are user
> adjustable.
>
> Just my 2c
>
>
> Rich
>
> Richard Cole
> Research Scientist V
> Director: Advanced Light Microscopy & Image Analysis Core
> Wadsworth Center
> P.O. Box 509 Albany N.Y. 12201-0509
>
> Research Assistant Professor
> Dept. of Biomedical Sciences
> School of Public Health State University of New York Albany, New York
>
> SS518-474-7048
> �{ �n518-474-4430
>   [hidden email]
> Website www.wadsworth.org/cores/alm/index.htm
>
>
> ________________________________________
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of CONFOCALMICROSCOPY automatic digest system
> Sent: Sunday, April 12, 2009 1:05 AM
> To: [hidden email]
> Subject: CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
>
>
>
>
>
> CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
> Table of contents:
> • Spectral: Single channel vs Multi-channel
> 1. Spectral: Single channel vs Multi-channel
> o Spectral: Single channel vs Multi-channel (04/11)
> From: "Richard E. Edelmann" <[hidden email]>
>
>
> Browse the CONFOCALMICROSCOPY online archives.
>
>
>
>
>
> IMPORTANT NOTICE: This e-mail and any attachments may contain
> confidential or sensitive information which is, or may be, legally
> privileged or otherwise protected by law from further disclosure.  It
> is intended only for the addressee.  If you received this in error or
> from someone who was not authorized to send it to you, please do not
> distribute, copy or use it or any attachments.  Please notify the
> sender immediately by reply e-mail and delete this from your
> system. Thank you for your cooperation.
>

[Badri Roysam]

Craig, Perhaps I worded my question incorrectly. I meant to ask about the
currently best achievable level of multiplexing.


Badri Roysam
Professor, Department of Electrical, Computer and Systems Engineering
Associate Director, NSF Center for Subsurface Sensing & Imaging Systems (CenSSIS ERC)
Co-Director, Rensselaer Center for Open Source Software
Rensselaer Polytechnic Institute
110 8th Street, Troy, New York 12180-3590, USA.
Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308): 518-276-8207, Fax: [phone]
Email: [hidden email], Web: http://www.ecse.rpi.edu/~roysam



----- Original Message -----
From: Craig Brideau [mailto:[hidden email]]
To: [hidden email]
Subject: Re: Spectral: Single channel vs Multi-channel

> The Nikon C1Si will do that with spectral unmixing.  Speed is about ~1fps
> @512x512 pixels.
>
> Craig
>
>
> On Mon, Apr 13, 2009 at 6:55 AM, Badri Roysam <[hidden email]> wrote:
>
> > While on this topic, I am always to eager to learn about ways to image
> many
> > more
> > channels at a time. Has anyone made any breakthroughs lately that may
> > permit us to
> > stain and image, say 10 - 20 channels...?
> >
> >
> > Badri Roysam
> > Professor, Department of Electrical, Computer and Systems Engineering
> > Associate Director, NSF Center for Subsurface Sensing & Imaging Systems
> > (CenSSIS ERC)
> > Co-Director, Rensselaer Center for Open Source Software
> > Rensselaer Polytechnic Institute
> > 110 8th Street, Troy, New York 12180-3590, USA.
> > Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308):
> > 518-276-8207, Fax: [phone]
> > Email: [hidden email], Web:
> http://www.ecse.rpi.edu/~roysam<http://www.ecse.rpi.edu/%7Eroysam>
> >
> >
> >
> > ----- Original Message -----
> > From: Richard Cole [mailto:[hidden email]]
> > To: [hidden email]
> > Subject: Spectral: Single channel vs Multi-channel
> >
> >
> > > I looked very carefully at both systems and my conclusion are:
> > >
> > > If you need speed a single channel, a multi-channel spectral PMT is the
> > way
> > > to go.
> > >
> > > If speed is not an issue, as I my case, the scanning system offers a
> > couple
> > > of major advantages
> > >
> > > 1. For spectral unmixing the dyes can have greatly different fluorescent
> > > levels (I have separated two over lapping fluorophores with 10X
> > > concentration difference)
> > >
> > > 2. The size of the slit and the number of sampling points are user
> > > adjustable.
> > >
> > > Just my 2c
> > >
> > >
> > > Rich
> > >
> > > Richard Cole
> > > Research Scientist V
> > > Director: Advanced Light Microscopy & Image Analysis Core
> > > Wadsworth Center
> > > P.O. Box 509 Albany N.Y. 12201-0509
> > >
> > > Research Assistant Professor
> > > Dept. of Biomedical Sciences
> > > School of Public Health State University of New York Albany, New York
> > >
> > > SS518-474-7048
> > > { n518-474-4430
> > >   [hidden email]
> > > Website www.wadsworth.org/cores/alm/index.htm
> > >
> > >
> > > ________________________________________
> > > From: Confocal Microscopy List [mailto:[hidden email]]
> > On
> > > Behalf Of CONFOCALMICROSCOPY automatic digest system
> > > Sent: Sunday, April 12, 2009 1:05 AM
> > > To: [hidden email]
> > > Subject: CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009
> > (#2009-72)
> > >
> > >
> > >
> > >
> > >
> > > CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
> > > Table of contents:
> > > * Spectral: Single channel vs Multi-channel
> > > 1. Spectral: Single channel vs Multi-channel
> > > o Spectral: Single channel vs Multi-channel (04/11)
> > > From: "Richard E. Edelmann" <[hidden email]>
> > >
> > >
> > > Browse the CONFOCALMICROSCOPY online archives.
> > >
> > >
> > >
> > >
> > >
> > > IMPORTANT NOTICE: This e-mail and any attachments may contain
> > > confidential or sensitive information which is, or may be, legally
> > > privileged or otherwise protected by law from further disclosure.  It
> > > is intended only for the addressee.  If you received this in error or
> > > from someone who was not authorized to send it to you, please do not
> > > distribute, copy or use it or any attachments.  Please notify the
> > > sender immediately by reply e-mail and delete this from your
> > > system. Thank you for your cooperation.
> > >
> >
>

[Craig Brideau]

Is your question: "What is the largest number of dyes that can practically be used with spectral unmixing?"
The current state of the art is really only limited by computing power.  The C1Si can handle up to eight (I think) or more dyes simultaneously and display the results in near-realtime.  We have our own software (ImageTrak) that we run on the data sets afterwards which is theoretically unlimited in the number of dyes it will unmix.  It is mainly limited by computing power and the time you are willing to spend to perform unmixes of multiple dyes.  More dyes = longer unmixing process.  In terms of accuracy we have unmixed dyes that are quite similar, for instance YFP and GFP, with perfect results.  Nikon has a sample slide with dyes that are even closer that their system and algorithm can reliably unmix.

Craig

On Tue, Apr 14, 2009 at 4:17 PM, Badri Roysam <[hidden email]> wrote:

Craig, Perhaps I worded my question incorrectly. I meant to ask about the
currently best achievable level of multiplexing.

Badri Roysam
Professor, Department of Electrical, Computer and Systems Engineering
Associate Director, NSF Center for Subsurface Sensing & Imaging Systems (CenSSIS ERC)
Co-Director, Rensselaer Center for Open Source Software
Rensselaer Polytechnic Institute
110 8th Street, Troy, New York 12180-3590, USA.
Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308): 518-276-8207, Fax: [phone]
Email: [hidden email], Web: http://www.ecse.rpi.edu/~roysam



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

From: Craig Brideau [mailto:[hidden email]]
To: [hidden email]

Subject: Re: Spectral: Single channel vs Multi-channel


> The Nikon C1Si will do that with spectral unmixing.  Speed is about ~1fps
> @512x512 pixels.
>
> Craig
>
>
> On Mon, Apr 13, 2009 at 6:55 AM, Badri Roysam <[hidden email]> wrote:
>
> > While on this topic, I am always to eager to learn about ways to image
> many
> > more
> > channels at a time. Has anyone made any breakthroughs lately that may
> > permit us to
> > stain and image, say 10 - 20 channels...?
> >
> >
> > Badri Roysam
> > Professor, Department of Electrical, Computer and Systems Engineering
> > Associate Director, NSF Center for Subsurface Sensing & Imaging Systems
> > (CenSSIS ERC)
> > Co-Director, Rensselaer Center for Open Source Software
> > Rensselaer Polytechnic Institute
> > 110 8th Street, Troy, New York 12180-3590, USA.
> > Office(JEC 7010): 518-276-8067, Assistant: 518-276-8525, Lab(JEC 6308):
> > 518-276-8207, Fax: [phone]
> > Email: [hidden email], Web:

> http://www.ecse.rpi.edu/~roysam<http://www.ecse.rpi.edu/%7Eroysam>

> >
> >
> >
> > ----- Original Message -----
> > From: Richard Cole [mailto:[hidden email]]
> > To: [hidden email]
> > Subject: Spectral: Single channel vs Multi-channel
> >
> >
> > > I looked very carefully at both systems and my conclusion are:
> > >
> > > If you need speed a single channel, a multi-channel spectral PMT is the
> > way
> > > to go.
> > >
> > > If speed is not an issue, as I my case, the scanning system offers a
> > couple
> > > of major advantages
> > >
> > > 1. For spectral unmixing the dyes can have greatly different fluorescent
> > > levels (I have separated two over lapping fluorophores with 10X
> > > concentration difference)
> > >
> > > 2. The size of the slit and the number of sampling points are user
> > > adjustable.
> > >
> > > Just my 2c
> > >
> > >
> > > Rich
> > >
> > > Richard Cole
> > > Research Scientist V
> > > Director: Advanced Light Microscopy & Image Analysis Core
> > > Wadsworth Center
> > > P.O. Box 509 Albany N.Y. 12201-0509
> > >
> > > Research Assistant Professor
> > > Dept. of Biomedical Sciences
> > > School of Public Health State University of New York Albany, New York
> > >
> > > SS518-474-7048
> > > { n518-474-4430
> > >   [hidden email]
> > > Website www.wadsworth.org/cores/alm/index.htm
> > >
> > >
> > > ________________________________________
> > > From: Confocal Microscopy List [mailto:[hidden email]]
> > On
> > > Behalf Of CONFOCALMICROSCOPY automatic digest system
> > > Sent: Sunday, April 12, 2009 1:05 AM
> > > To: [hidden email]
> > > Subject: CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009
> > (#2009-72)
> > >
> > >
> > >
> > >
> > >
> > > CONFOCALMICROSCOPY Digest - 10 Apr 2009 to 11 Apr 2009 (#2009-72)
> > > Table of contents:
> > > * Spectral: Single channel vs Multi-channel
> > > 1. Spectral: Single channel vs Multi-channel
> > > o Spectral: Single channel vs Multi-channel (04/11)
> > > From: "Richard E. Edelmann" <[hidden email]>
> > >
> > >
> > > Browse the CONFOCALMICROSCOPY online archives.
> > >
> > >
> > >
> > >
> > >
> > > IMPORTANT NOTICE: This e-mail and any attachments may contain
> > > confidential or sensitive information which is, or may be, legally
> > > privileged or otherwise protected by law from further disclosure.  It
> > > is intended only for the addressee.  If you received this in error or
> > > from someone who was not authorized to send it to you, please do not
> > > distribute, copy or use it or any attachments.  Please notify the
> > > sender immediately by reply e-mail and delete this from your
> > > system. Thank you for your cooperation.
> > >
> >
>

[Alessandro Esposito]

Dear Richard,
   I guess it depends a lot on how many spectral channels you need. We
probably wish to have perfect high resolution spectra, but often they are not
really needed unless you wish to analyze the "fine" structures of spectra.
Would you make unmixing, then the number of channels and required resolution
will be somehow related to the number of fluorophores to be unmixed and their
minimal separation.

This preamble to say that a prism-based system such the Leica SP* equipped
with the 2 (if I remember correctly) additional spectral channels already
provide high photon-efficiency and 5 spectral channels. The lambda scan
option of the Leica is quite slow and therefore useful only for photostable and
usually fixed samples (also be aware of some non-linearities of the detection
system which will "deform" the spectra).

The spectral options of Nikon and Zeiss will be somehow less photon efficient
compared to the collection of 3-5 parallel channels with a Leica, but certainly
comparatevly faster and more photon efficient than scanning a higher number
of channels.

We are using a prism based EMCCD spectrograph for spectral detection
coupled with a confocal getting 128 spectral channels with pixel dwell times of
100-400 us). This allows to combine the high efficiency of prism based
detection with the high quantum efficiencies of EMCCDs. I guess that if you
need a high number of channels this would be the optimal solution, but often
fewer channels already do the job.

Regards,

Alessandro Esposito
Laser Analytics Group
Dept. Chemical Engineering and Biotechnology
University of Cambridge