Confocal Microscopy List

Imaging near-IR dyes by confocal

Classic

List

Threaded

10 messages Options

Wendy Salmon

Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello all,

I am hoping to get a community feeling for demand and implementation of imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over 740nm), using confocal. I am seeing more and more requests from researchers wanting to image these probes in order to correlate whole-animal imaging with sub-cellular localization.

What system(s) are capable of this, either commercial or home-built? Based on a previous thread (link below) it sounds like SDC with EMCCD could do it. However, are the optics in the Yokogawa head capable of transmitting the near-IR excitation and emission light without massive aberration? Additionally, a point scanning option would be helpful for samples that are thick and heavily stained--are there any reasonable options for this?

Currently I recommend researcher tag their probe with two fluorophores for the different applications--one near-IR for the whole animal and one in the visible wavelengths for the confocal--but there are concerns about fixation quenching, autofluorescence, binding chemistry, etc.

Many thanks!
Wendy

Previous thread related to near-IR dyes:
http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477

~~~~~~~~~~~~~~~~~~~~~~~
Wendy Salmon
Light Microscopy Specialist
Whitehead Institute for Biomedical Research
W.M. Keck Imaging Facility
9 Cambridge Center, Rm 447
Cambridge, MA 02142
c: 617-429-0158
e: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/

Armstrong, Brian

Re: Imaging near-IR dyes by confocal

Hi Wendy, I also get requests to image IR dyes periodically.
I just ordered a new Confocal and was somewhat surprised that the highest LASER was ~639nm. These IR dyes are becoming ubiquitous in other cytometry techniques so it seems LASER-scanning Confocal microscopes are falling behind in this respect.
It certainly seems that IR dyes will be in our future.

Cheers,

Brian D Armstrong PhD
Associate Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Wendy Salmon
Sent: Wednesday, February 10, 2016 3:01 PM
To: [hidden email]
Subject: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello all,

I am hoping to get a community feeling for demand and implementation of imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over 740nm), using confocal. I am seeing more and more requests from researchers wanting to image these probes in order to correlate whole-animal imaging with sub-cellular localization.

What system(s) are capable of this, either commercial or home-built? Based on a previous thread (link below) it sounds like SDC with EMCCD could do it. However, are the optics in the Yokogawa head capable of transmitting the near-IR excitation and emission light without massive aberration? Additionally, a point scanning option would be helpful for samples that are thick and heavily stained--are there any reasonable options for this?

Currently I recommend researcher tag their probe with two fluorophores for the different applications--one near-IR for the whole animal and one in the visible wavelengths for the confocal--but there are concerns about fixation quenching, autofluorescence, binding chemistry, etc.

Many thanks!
Wendy

Previous thread related to near-IR dyes:
http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477

~~~~~~~~~~~~~~~~~~~~~~~
Wendy Salmon
Light Microscopy Specialist
Whitehead Institute for Biomedical Research
W.M. Keck Imaging Facility
9 Cambridge Center, Rm 447
Cambridge, MA 02142
c: 617-429-0158
e: [hidden email]
w: http://staffa.wi.mit.edu/microscopy/

---------------------------------------------------------------------
*SECURITY/CONFIDENTIALITY WARNING:
This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p)
---------------------------------------------------------------------

Craig Brideau

Re: Imaging near-IR dyes by confocal

In reply to this post by Wendy Salmon

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

A key limitation is detector sensitivity at longer wavelengths. GaAsP
detector sensitivity falls off very rapidly in the red wavelength ranges.
You are actually better off with a standard multi-alkali PMT (-40 type
hamamatsu) than a GaAsP detector if you are trying to detect wavelengths
longer than ~650nm. Regular bi-alkali PMTs are also not super-great at long
wavelengths either; multi-alkali is best.

Craig

On Wed, Feb 10, 2016 at 4:01 PM, Wendy Salmon <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello all,
>
> I am hoping to get a community feeling for demand and implementation of
> imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over
> 740nm), using confocal. I am seeing more and more requests from researchers
> wanting to image these probes in order to correlate whole-animal imaging
> with sub-cellular localization.
>
> What system(s) are capable of this, either commercial or home-built? Based
> on a previous thread (link below) it sounds like SDC with EMCCD could do
> it. However, are the optics in the Yokogawa head capable of transmitting
> the near-IR excitation and emission light without massive aberration?
> Additionally, a point scanning option would be helpful for samples that are
> thick and heavily stained--are there any reasonable options for this?
>
> Currently I recommend researcher tag their probe with two fluorophores for
> the different applications--one near-IR for the whole animal and one in the
> visible wavelengths for the confocal--but there are concerns about fixation
> quenching, autofluorescence, binding chemistry, etc.
>
> Many thanks!
> Wendy
>
> Previous thread related to near-IR dyes:
> http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477
>
> ~~~~~~~~~~~~~~~~~~~~~~~
> Wendy Salmon
> Light Microscopy Specialist
> Whitehead Institute for Biomedical Research
> W.M. Keck Imaging Facility
> 9 Cambridge Center, Rm 447
> Cambridge, MA 02142
> c: 617-429-0158
> e: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/
>

Julio Vazquez-2

Re: Imaging near-IR dyes by confocal

In reply to this post by Armstrong, Brian

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I don't know about point scanners, but for widefield systems, some vendors may be catching up. For example, The Cellomics Arrayscan high content reader now offers a 740 nm excitation line and matching emission band. Andor have a note on their web site about imaging with 730 nm Ex/780nm Em. I think they may offer this option on their spinning disk system. Lasers for these applications are available (our Licor OdysseyNIR scanner uses 680 and 780 nm ); sCMOS cameras such as Orca FLASH 4 v2 have QE around 50% at 800 nm, and the Yokogawa CSU-W1 scanner has optional ports for NIR lasers (685/785).

http://www.andor.com/learning-academy/why-image-with-near-infrared-wavelengths-application-note-on-borealis-perfect-illumination-delivery™

http://www.yokogawa.com/scanner/product/csu/csuw1_3_e.htm

Julio Vazquez
Fred Hutchinson Cancer Research Center.
--

On Feb 10, 2016, at 3:31 PM, Armstrong, Brian wrote:

> Hi Wendy, I also get requests to image IR dyes periodically.
> I just ordered a new Confocal and was somewhat surprised that the highest LASER was ~639nm. These IR dyes are becoming ubiquitous in other cytometry techniques so it seems LASER-scanning Confocal microscopes are falling behind in this respect.
> It certainly seems that IR dyes will be in our future.
>
> Cheers,
>
> Brian D Armstrong PhD
> Associate Research Professor
> Director, Light Microscopy Core
> Beckman Research Institute
> City of Hope
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Wendy Salmon
> Sent: Wednesday, February 10, 2016 3:01 PM
> To: [hidden email]
> Subject: Imaging near-IR dyes by confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello all,
>
> I am hoping to get a community feeling for demand and implementation of imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over 740nm), using confocal. I am seeing more and more requests from researchers wanting to image these probes in order to correlate whole-animal imaging with sub-cellular localization.
>
> What system(s) are capable of this, either commercial or home-built? Based on a previous thread (link below) it sounds like SDC with EMCCD could do it. However, are the optics in the Yokogawa head capable of transmitting the near-IR excitation and emission light without massive aberration? Additionally, a point scanning option would be helpful for samples that are thick and heavily stained--are there any reasonable options for this?
>
> Currently I recommend researcher tag their probe with two fluorophores for the different applications--one near-IR for the whole animal and one in the visible wavelengths for the confocal--but there are concerns about fixation quenching, autofluorescence, binding chemistry, etc.
>
> Many thanks!
> Wendy
>
> Previous thread related to near-IR dyes:
> http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477
>
> ~~~~~~~~~~~~~~~~~~~~~~~
> Wendy Salmon
> Light Microscopy Specialist
> Whitehead Institute for Biomedical Research
> W.M. Keck Imaging Facility
> 9 Cambridge Center, Rm 447
> Cambridge, MA 02142
> c: 617-429-0158
> e: [hidden email]
> w: http://staffa.wi.mit.edu/microscopy/
>
>
> ---------------------------------------------------------------------
> *SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p)
> ---------------------------------------------------------------------
>

Sripad Ram-2

Re: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Wendy,
Some confocal vendors do provide custom (aka special order) solution for
NIR sensitive PMT that will detect upto 900 nm. In addition you'd would
also require NIR laser (740 and/or 785 nm). Please note that the typical QE
of the PMT will be quite low (around 5% so it will work for bightly stained
samples.

Sripad

On Wed, Feb 10, 2016 at 4:48 PM, Julio Vazquez <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> I don't know about point scanners, but for widefield systems, some vendors
> may be catching up. For example, The Cellomics Arrayscan high content
> reader now offers a 740 nm excitation line and matching emission band.
> Andor have a note on their web site about imaging with 730 nm Ex/780nm Em.
> I think they may offer this option on their spinning disk system. Lasers
> for these applications are available (our Licor OdysseyNIR scanner uses 680
> and 780 nm ); sCMOS cameras such as Orca FLASH 4 v2 have QE around 50% at
> 800 nm, and the Yokogawa CSU-W1 scanner has optional ports for NIR lasers
> (685/785).
>
>
> http://www.andor.com/learning-academy/why-image-with-near-infrared-wavelengths-application-note-on-borealis-perfect-illumination-delivery
> ™
>
> http://www.yokogawa.com/scanner/product/csu/csuw1_3_e.htm
>
>
> Julio Vazquez
> Fred Hutchinson Cancer Research Center.
> --
>
> On Feb 10, 2016, at 3:31 PM, Armstrong, Brian wrote:
>
> > Hi Wendy, I also get requests to image IR dyes periodically.
> > I just ordered a new Confocal and was somewhat surprised that the
> highest LASER was ~639nm. These IR dyes are becoming ubiquitous in other
> cytometry techniques so it seems LASER-scanning Confocal microscopes are
> falling behind in this respect.
> > It certainly seems that IR dyes will be in our future.
> >
> > Cheers,
> >
> > Brian D Armstrong PhD
> > Associate Research Professor
> > Director, Light Microscopy Core
> > Beckman Research Institute
> > City of Hope
> > 1450 E Duarte Rd
> > Duarte, CA 91010
> > 626-256-4673 x62872
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Wendy Salmon
> > Sent: Wednesday, February 10, 2016 3:01 PM
> > To: [hidden email]
> > Subject: Imaging near-IR dyes by confocal
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hello all,
> >
> > I am hoping to get a community feeling for demand and implementation of
> imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over
> 740nm), using confocal. I am seeing more and more requests from researchers
> wanting to image these probes in order to correlate whole-animal imaging
> with sub-cellular localization.
> >
> > What system(s) are capable of this, either commercial or home-built?
> Based on a previous thread (link below) it sounds like SDC with EMCCD could
> do it. However, are the optics in the Yokogawa head capable of transmitting
> the near-IR excitation and emission light without massive aberration?
> Additionally, a point scanning option would be helpful for samples that are
> thick and heavily stained--are there any reasonable options for this?
> >
> > Currently I recommend researcher tag their probe with two fluorophores
> for the different applications--one near-IR for the whole animal and one in
> the visible wavelengths for the confocal--but there are concerns about
> fixation quenching, autofluorescence, binding chemistry, etc.
> >
> > Many thanks!
> > Wendy
> >
> > Previous thread related to near-IR dyes:
> > http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477
> >
> > ~~~~~~~~~~~~~~~~~~~~~~~
> > Wendy Salmon
> > Light Microscopy Specialist
> > Whitehead Institute for Biomedical Research
> > W.M. Keck Imaging Facility
> > 9 Cambridge Center, Rm 447
> > Cambridge, MA 02142
> > c: 617-429-0158
> > e: [hidden email]
> > w: http://staffa.wi.mit.edu/microscopy/
> >
> >
> > ---------------------------------------------------------------------
> > *SECURITY/CONFIDENTIALITY WARNING:
> > This message and any attachments are intended solely for the individual
> or entity to which they are addressed. This communication may contain
> information that is privileged, confidential, or exempt from disclosure
> under applicable law (e.g., personal health information, research data,
> financial information). Because this e-mail has been sent without
> encryption, individuals other than the intended recipient may be able to
> view the information, forward it to others or tamper with the information
> without the knowledge or consent of the sender. If you are not the intended
> recipient, or the employee or person responsible for delivering the message
> to the intended recipient, any dissemination, distribution or copying of
> the communication is strictly prohibited. If you received the communication
> in error, please notify the sender immediately by replying to this message
> and deleting the message and any accompanying files from your system. If,
> due to the security risks, you do not wish to receive further
> communications via e-mail, please reply to this message and inform the
> sender that you do not wish to receive further e-mail from the sender.
> (fpc5p)
> > ---------------------------------------------------------------------
> >
>

Schwartz, Owen (NIH/NIAID) [E]

Re: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello all,
Leica offers a 690 nm CW laser for the SP8 in addition to the standard set of lasers. It works great for Alexa 700 etc. we have also worked with dyes that have emission in the mid 800s but use APDs for detection. This has been very successful.

Owen M Schwartz
NIAID-NIH
Sent from my iPhone

> On Feb 10, 2016, at 8:09 PM, S Ram <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Wendy,
> Some confocal vendors do provide custom (aka special order) solution for
> NIR sensitive PMT that will detect upto 900 nm. In addition you'd would
> also require NIR laser (740 and/or 785 nm). Please note that the typical QE
> of the PMT will be quite low (around 5% so it will work for bightly stained
> samples.
>
> Sripad
>
>
> On Wed, Feb 10, 2016 at 4:48 PM, Julio Vazquez <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> I don't know about point scanners, but for widefield systems, some vendors
>> may be catching up. For example, The Cellomics Arrayscan high content
>> reader now offers a 740 nm excitation line and matching emission band.
>> Andor have a note on their web site about imaging with 730 nm Ex/780nm Em.
>> I think they may offer this option on their spinning disk system. Lasers
>> for these applications are available (our Licor OdysseyNIR scanner uses 680
>> and 780 nm ); sCMOS cameras such as Orca FLASH 4 v2 have QE around 50% at
>> 800 nm, and the Yokogawa CSU-W1 scanner has optional ports for NIR lasers
>> (685/785).
>>
>>
>> http://www.andor.com/learning-academy/why-image-with-near-infrared-wavelengths-application-note-on-borealis-perfect-illumination-delivery
>> ™
>>
>> http://www.yokogawa.com/scanner/product/csu/csuw1_3_e.htm
>>
>>
>> Julio Vazquez
>> Fred Hutchinson Cancer Research Center.
>> --
>>
>>> On Feb 10, 2016, at 3:31 PM, Armstrong, Brian wrote:
>>>
>>> Hi Wendy, I also get requests to image IR dyes periodically.
>>> I just ordered a new Confocal and was somewhat surprised that the
>> highest LASER was ~639nm. These IR dyes are becoming ubiquitous in other
>> cytometry techniques so it seems LASER-scanning Confocal microscopes are
>> falling behind in this respect.
>>> It certainly seems that IR dyes will be in our future.
>>>
>>> Cheers,
>>>
>>> Brian D Armstrong PhD
>>> Associate Research Professor
>>> Director, Light Microscopy Core
>>> Beckman Research Institute
>>> City of Hope
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Wendy Salmon
>>> Sent: Wednesday, February 10, 2016 3:01 PM
>>> To: [hidden email]
>>> Subject: Imaging near-IR dyes by confocal
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hello all,
>>>
>>> I am hoping to get a community feeling for demand and implementation of
>> imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission over
>> 740nm), using confocal. I am seeing more and more requests from researchers
>> wanting to image these probes in order to correlate whole-animal imaging
>> with sub-cellular localization.
>>>
>>> What system(s) are capable of this, either commercial or home-built?
>> Based on a previous thread (link below) it sounds like SDC with EMCCD could
>> do it. However, are the optics in the Yokogawa head capable of transmitting
>> the near-IR excitation and emission light without massive aberration?
>> Additionally, a point scanning option would be helpful for samples that are
>> thick and heavily stained--are there any reasonable options for this?
>>>
>>> Currently I recommend researcher tag their probe with two fluorophores
>> for the different applications--one near-IR for the whole animal and one in
>> the visible wavelengths for the confocal--but there are concerns about
>> fixation quenching, autofluorescence, binding chemistry, etc.
>>>
>>> Many thanks!
>>> Wendy
>>>
>>> Previous thread related to near-IR dyes:
>>> http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5477
>>>
>>> ~~~~~~~~~~~~~~~~~~~~~~~
>>> Wendy Salmon
>>> Light Microscopy Specialist
>>> Whitehead Institute for Biomedical Research
>>> W.M. Keck Imaging Facility
>>> 9 Cambridge Center, Rm 447
>>> Cambridge, MA 02142
>>> c: 617-429-0158
>>> e: [hidden email]
>>> w: http://staffa.wi.mit.edu/microscopy/
>>>
>>>
>>> ---------------------------------------------------------------------
>>> *SECURITY/CONFIDENTIALITY WARNING:
>>> This message and any attachments are intended solely for the individual
>> or entity to which they are addressed. This communication may contain
>> information that is privileged, confidential, or exempt from disclosure
>> under applicable law (e.g., personal health information, research data,
>> financial information). Because this e-mail has been sent without
>> encryption, individuals other than the intended recipient may be able to
>> view the information, forward it to others or tamper with the information
>> without the knowledge or consent of the sender. If you are not the intended
>> recipient, or the employee or person responsible for delivering the message
>> to the intended recipient, any dissemination, distribution or copying of
>> the communication is strictly prohibited. If you received the communication
>> in error, please notify the sender immediately by replying to this message
>> and deleting the message and any accompanying files from your system. If,
>> due to the security risks, you do not wish to receive further
>> communications via e-mail, please reply to this message and inform the
>> sender that you do not wish to receive further e-mail from the sender.
>> (fpc5p)
>>> ---------------------------------------------------------------------
>>

Vitaly Boyko

Re: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Wendy,
On Leica SP8 we use 670 nm line of the White Light Laser and can easily separate Alexa647/Cy5 from the Li-Cor IR 800 or other NIR dyes. We use Perkin Elmer APDs for the detection of tdTomato, mCherry, iRFP, Alexa647, Alexa 750, Alexa 790, Cy7, ir683, ir800 etc. Works well. If you wish, you can order APDs (through Leica), now manufactured by Excelitas Technologies in Canada. I would suggest APDs with lower noise (less than 2000 counts per second). Zeiss, unfortunately does not offer White Light Laser, but you may upgrade Zeiss confocals with the Excelitas APDs. Otherwise, Leica SP8 is great system, if you have funds to add Leica SP8 to your Zeiss portfolio. Leica Tech Service is very good in the US - with much lower number of direct calls to Germany (Zeiss Techs in the US often get "lost" without direct German supervision, sorry for the plain "half-joke").
I am ready to share more details on NIR imaging, please contact me off-line.I am rarely check my Yahoo e-mail box. Please find me via LinkedIn or via regularly checked e-mail - [hidden email] Vitaly

On Wednesday, February 10, 2016 9:15 PM, "Schwartz, Owen (NIH/NIAID) [E]" <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello all,
Leica offers a 690 nm CW laser for the SP8 in addition to the standard set of lasers. It works great for Alexa 700 etc. we have also worked with dyes that have emission in the mid 800s but use APDs for detection. This has been very successful.

Owen M Schwartz
NIAID-NIH
Sent from my iPhone

WAINWRIGHT James

Re: Imaging near-IR dyes by confocal

*** COMMERCIAL RESPONSE ***

We have installed a number of NIR-capable (e.g. 730 nm laser) spinning disk systems.

Both EMCCD and sCMOS maintain good QE out into the NIR region (~70% and ~50% respectively @ 800nm).

One thing to bear in mind with any NIR system is to make sure that your microscope does not have any filters or dichroics that may interfere and cannot be removed (e.g. some motorised "auto" focus / anti-drift mechanisms). It would be a good idea to discuss this with your microscope vendor and to plan a specific stand or ensure that your existing (or planned) stand can transmit the desired emission wavelengths.

Best wishes,

James

James Wainwright
Product Support Engineer (Microscopy Systems)
Europe, Middle East & Africa

http://www.Andor.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vitaly Boyko
Sent: 11 February 2016 03:33
To: [hidden email]
Subject: Re: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Wendy,
On Leica SP8 we use 670 nm line of the White Light Laser and can easily separate Alexa647/Cy5 from the Li-Cor IR 800 or other NIR dyes. We use Perkin Elmer APDs for the detection of tdTomato, mCherry, iRFP, Alexa647, Alexa 750, Alexa 790, Cy7, ir683, ir800 etc. Works well. If you wish, you can order APDs (through Leica), now manufactured by Excelitas Technologies in Canada. I would suggest APDs with lower noise (less than 2000 counts per second). Zeiss, unfortunately does not offer White Light Laser, but you may upgrade Zeiss confocals with the Excelitas APDs. Otherwise, Leica SP8 is great system, if you have funds to add Leica SP8 to your Zeiss portfolio. Leica Tech Service is very good in the US - with much lower number of direct calls to Germany (Zeiss Techs in the US often get "lost" without direct German supervision, sorry for the plain "half-joke"). I am ready to share more details on NIR imaging, please contact me off-line.I am rarely check my Yahoo e-mail box. Please find me via LinkedIn or via regularly checked e-mail - [hidden email] Vitaly

On Wednesday, February 10, 2016 9:15 PM, "Schwartz, Owen (NIH/NIAID) [E]" <[hidden email]> wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hello all,
Leica offers a 690 nm CW laser for the SP8 in addition to the standard set of lasers. It works great for Alexa 700 etc. we have also worked with dyes that have emission in the mid 800s but use APDs for detection. This has been very successful.

Owen M Schwartz
NIAID-NIH
Sent from my iPhone

> On Feb 10, 2016, at 8:09 PM, S Ram <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Wendy,
> Some confocal vendors do provide custom (aka special order) solution
> for NIR sensitive PMT that will detect upto 900 nm. In addition you'd
> would also require NIR laser (740 and/or 785 nm). Please note that the
> typical QE of the PMT will be quite low (around 5% so it will work for
> bightly stained samples.
>
> Sripad
>
>
> On Wed, Feb 10, 2016 at 4:48 PM, Julio Vazquez
> <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> I don't know about point scanners, but for widefield systems, some
>> vendors may be catching up. For example, The Cellomics Arrayscan high
>> content reader now offers a 740 nm excitation line and matching emission band.
>> Andor have a note on their web site about imaging with 730 nm Ex/780nm Em.
>> I think they may offer this option on their spinning disk system.
>> Lasers for these applications are available (our Licor OdysseyNIR
>> scanner uses 680 and 780 nm ); sCMOS cameras such as Orca FLASH 4 v2
>> have QE around 50% at
>> 800 nm, and the Yokogawa CSU-W1 scanner has optional ports for NIR
>> lasers (685/785).
>>
>>
>> http://www.andor.com/learning-academy/why-image-with-near-infrared-wa
>> velengths-application-note-on-borealis-perfect-illumination-delivery
>> ™
>>
>> http://www.yokogawa.com/scanner/product/csu/csuw1_3_e.htm
>>
>>
>> Julio Vazquez
>> Fred Hutchinson Cancer Research Center.
>> --
>>
>>> On Feb 10, 2016, at 3:31 PM, Armstrong, Brian wrote:
>>>
>>> Hi Wendy, I also get requests to image IR dyes periodically.
>>> I just ordered a new Confocal and was somewhat surprised that the
>> highest LASER was ~639nm. These IR dyes are becoming ubiquitous in
>> other cytometry techniques so it seems LASER-scanning Confocal
>> microscopes are falling behind in this respect.
>>> It certainly seems that IR dyes will be in our future.
>>>
>>> Cheers,
>>>
>>> Brian D Armstrong PhD
>>> Associate Research Professor
>>> Director, Light Microscopy Core
>>> Beckman Research Institute
>>> City of Hope
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]]
>> On Behalf Of Wendy Salmon
>>> Sent: Wednesday, February 10, 2016 3:01 PM
>>> To: [hidden email]
>>> Subject: Imaging near-IR dyes by confocal
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hello all,
>>>
>>> I am hoping to get a community feeling for demand and implementation
>>> of
>> imaging near-IR dyes such as Cy7, iRFP and carbon nanotubes (emission
>> over 740nm), using confocal. I am seeing more and more requests from
>> researchers wanting to image these probes in order to correlate
>> whole-animal imaging with sub-cellular localization.
>>>
>>> What system(s) are capable of this, either commercial or home-built?
>> Based on a previous thread (link below) it sounds like SDC with EMCCD
>> could do it. However, are the optics in the Yokogawa head capable of
>> transmitting the near-IR excitation and emission light without massive aberration?
>> Additionally, a point scanning option would be helpful for samples
>> that are thick and heavily stained--are there any reasonable options for this?
>>>
>>> Currently I recommend researcher tag their probe with two
>>> fluorophores
>> for the different applications--one near-IR for the whole animal and
>> one in the visible wavelengths for the confocal--but there are
>> concerns about fixation quenching, autofluorescence, binding chemistry, etc.
>>>
>>> Many thanks!
>>> Wendy
>>>
>>> Previous thread related to near-IR dyes:
>>> http://lists.umn.edu/cgi-bin/wa?A2=ind1504&L=CONFOCALMICROSCOPY&P=R5
>>> 477
>>>
>>> ~~~~~~~~~~~~~~~~~~~~~~~
>>> Wendy Salmon
>>> Light Microscopy Specialist
>>> Whitehead Institute for Biomedical Research W.M. Keck Imaging
>>> Facility
>>> 9 Cambridge Center, Rm 447
>>> Cambridge, MA 02142
>>> c: 617-429-0158
>>> e: [hidden email]
>>> w: http://staffa.wi.mit.edu/microscopy/
>>>
>>>
>>> --------------------------------------------------------------------
>>> -
>>> *SECURITY/CONFIDENTIALITY WARNING:
>>> This message and any attachments are intended solely for the
>>> individual
>> or entity to which they are addressed. This communication may contain
>> information that is privileged, confidential, or exempt from
>> disclosure under applicable law (e.g., personal health information,
>> research data, financial information). Because this e-mail has been
>> sent without encryption, individuals other than the intended
>> recipient may be able to view the information, forward it to others
>> or tamper with the information without the knowledge or consent of
>> the sender. If you are not the intended recipient, or the employee or
>> person responsible for delivering the message to the intended
>> recipient, any dissemination, distribution or copying of the
>> communication is strictly prohibited. If you received the
>> communication in error, please notify the sender immediately by
>> replying to this message and deleting the message and any
>> accompanying files from your system. If, due to the security risks,
>> you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
>> (fpc5p)
>>> --------------------------------------------------------------------
>>> -
>>

This message has been scanned for malware by SurfControl plc. www.surfcontrol.com

Andrea Latini

Re: Imaging near-IR dyes by confocal

In reply to this post by Wendy Salmon

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

*** COMMERCIAL RESPONSE ***

dear Wendy,
I can confirm our confocals are normally used in a variety of OEM HCS and
HCA systems available on the market for NIR applications given the
customization we adopted on AR coatings of optical components (including
spinning disk and relay system).
The relay system is a custom design as well, diffraction limited over a FOV
of 18mm (diagonal).

Moreover, for a custom application we are testing a new confocal system
(based on our standard X-Light V1 & V2 design), for emission range
900nm-1600nm -
diffraction limited - and excitation range (lasers) 650nm-1000nm.
Detector, in this case, is Princeton Instruments NIRvana SWIR InGaAs array
with 900nm-1700nm sensitivity (>80% QE).

Best Regards.

Andrea Latini
President
CrestOptics
www.crestopt.com

yuansheng sun

Re: Imaging near-IR dyes by confocal

In reply to this post by Wendy Salmon

*** COMMERCIAL RESPONSE ***
Dear Wendy,
Last week, I was on one of our customers site. We tried two NIR dyes, af700 and af750 on our Alba system. Both worked well. I was using the spectraphysics maitai as the excitation source at 695nm and 745nm, and the excelitas spcm aqrh apds.
Earlier when i was in the lab, i also imaged nir dyes like ir783 with the Fianium supercontinous laser and also apds.
If you need, i can give more details off the list.
thanks, sheng

Sent from my T-Mobile 4G LTE Device

-------- Original message --------
From: Andrea Latini <[hidden email]>
Date: 2/11/2016 2:26 AM (GMT-08:00)
To: [hidden email]
Subject: Re: Imaging near-IR dyes by confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

*** COMMERCIAL RESPONSE ***

dear Wendy,
I can confirm our confocals are normally used in a variety of OEM HCS and
HCA systems available on the market for NIR applications given the
customization we adopted on AR coatings of optical components (including
spinning disk and relay system).
The relay system is a custom design as well, diffraction limited over a FOV
of 18mm (diagonal).

Moreover, for a custom application we are testing a new confocal system
(based on our standard X-Light V1 & V2 design), for emission range
900nm-1600nm -
diffraction limited - and excitation range (lasers) 650nm-1000nm.
Detector, in this case, is Princeton Instruments NIRvana SWIR InGaAs array
with 900nm-1700nm sensitivity (>80% QE).

Best Regards.

Andrea Latini
President
CrestOptics
www.crestopt.com