Infra-red confocal microscopy

[Claude Messier]

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I have been using Alexa 680 and Li-Cor IrDye800 for regular fluorescence
microscopy. I am trying to migrate infra-red dyes to confocal systems
particularly the single-photon variety using spectral detection with a
photomultiplier tube detection. Two problems are obvious the laser source
and the sensitivity of the photomultiplier tube in the infra-red region. Typical
photomultiplier tubes detector have low recovery when approaching 800nm
so that is a problem. There are infra-red diode lasers close enough to the
excitation peak of IrDye800 to be useable.
My question is: has anyone experimented with these infra-red dyes in
spectral detection system. Any information that would indicate that this is
possible would be appreciated - most important is whether PMT's can recover
enough photons at the infra-red end of the sensitivity.
Many thanks

[George McNamara]

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Hi Claude,

Li-Cor's Odyssey scanner uses APDs. Many FCS confocal microscopes use
APDs - but not necessarily the filter set(s) for fluorophores such as
IRDye800 (or more clinically, ICG). As for PMTs in the IR: if they have
low quantum efficiency but the specimen has practically no
autofluorescence (and no non-specific fluorescence of the NIRF), SNR may
approach #/0. On the other hand, infrachromatic [yes, this is a word]
correction may be poor (or good), depending on make and model of
microscope and especially objective lens -- not a big deal if you are
only looking at one fluorophore. Most back illuminated CCD's have nice
QE's in the NIR.

Enjoy,


George
p.s. while looking up infrachromatic on the Internet I came across    
http://iopscience.iop.org/0036-021X/66/3/R04    which reminded me that
multiphoton imaging has been around for well over a century. I wonder
whether more images were acquired that way or by cameras and PMTs, APDs
etc.  I am not going to trade in my Chameleon or APDs for a bucket of
supersensitizer, but figured worth mentioning at least one alternative
has existed for a lng time.


On 7/25/2011 10:19 PM, Claude Messier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have been using Alexa 680 and Li-Cor IrDye800 for regular fluorescence
> microscopy. I am trying to migrate infra-red dyes to confocal systems
> particularly the single-photon variety using spectral detection with a
> photomultiplier tube detection. Two problems are obvious the laser source
> and the sensitivity of the photomultiplier tube in the infra-red region. Typical
> photomultiplier tubes detector have low recovery when approaching 800nm
> so that is a problem. There are infra-red diode lasers close enough to the
> excitation peak of IrDye800 to be useable.
> My question is: has anyone experimented with these infra-red dyes in
> spectral detection system. Any information that would indicate that this is
> possible would be appreciated - most important is whether PMT's can recover
> enough photons at the infra-red end of the sensitivity.
> Many thanks
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Craig Brideau]

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Lenses designed for 2P microscopy might help here.  They tend to have more
IR correction.  Olympus' XLUMPLFLN or LUMP/IR water dipping lenses are an
example.  I think most manufacturers have similar lenses.  You will probably
need to tweak your collection optics however.  The good news is that PMTs
are fairly tolerant of beam focus differences...  The bad news is that your
confocal pinhole isn't, and for longer wavelengths you will need to open it
up anyway.

On a related note, I'm waiting for somebody to use a Cr:Forsterite laser to
do 2 photon with these NIR dyes.  Cr:Forsterite is similar to Ti:Saph, but
at 1200-1300nm so it can 2P excite dyes that are further into the NIR.  The
main limit right now seems to be in the optics.

Craig


On Mon, Jul 25, 2011 at 9:38 PM, George McNamara
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hi Claude,
>
> Li-Cor's Odyssey scanner uses APDs. Many FCS confocal microscopes use APDs
> - but not necessarily the filter set(s) for fluorophores such as IRDye800
> (or more clinically, ICG). As for PMTs in the IR: if they have low quantum
> efficiency but the specimen has practically no autofluorescence (and no
> non-specific fluorescence of the NIRF), SNR may approach #/0. On the other
> hand, infrachromatic [yes, this is a word] correction may be poor (or good),
> depending on make and model of microscope and especially objective lens --
> not a big deal if you are only looking at one fluorophore. Most back
> illuminated CCD's have nice QE's in the NIR.
>
> Enjoy,
>
>
> George
> p.s. while looking up infrachromatic on the Internet I came across
> http://iopscience.iop.org/**0036-021X/66/3/R04<http://iopscience.iop.org/0036-021X/66/3/R04>   which reminded me that multiphoton imaging has been around for well over
> a century. I wonder whether more images were acquired that way or by cameras
> and PMTs, APDs etc.  I am not going to trade in my Chameleon or APDs for a
> bucket of supersensitizer, but figured worth mentioning at least one
> alternative has existed for a lng time.
>
>
>
> On 7/25/2011 10:19 PM, Claude Messier wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> I have been using Alexa 680 and Li-Cor IrDye800 for regular fluorescence
>> microscopy. I am trying to migrate infra-red dyes to confocal systems
>> particularly the single-photon variety using spectral detection with a
>> photomultiplier tube detection. Two problems are obvious the laser source
>> and the sensitivity of the photomultiplier tube in the infra-red region.
>> Typical
>> photomultiplier tubes detector have low recovery when approaching 800nm
>> so that is a problem. There are infra-red diode lasers close enough to the
>> excitation peak of IrDye800 to be useable.
>> My question is: has anyone experimented with these infra-red dyes in
>> spectral detection system. Any information that would indicate that this
>> is
>> possible would be appreciated - most important is whether PMT's can
>> recover
>> enough photons at the infra-red end of the sensitivity.
>> Many thanks
>>
>>
>>
>
>
> --
>
>
> George McNamara, PhD
> Analytical Imaging Core Facility
> University of Miami
>

[Dan Stevens-2]

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I am similarly interested in the possibility of IR imaging (700-900nm) on
traditional PMTs.

I have had some feedback that imaging QDots at ~750 emission was
unsuccessful.  The PMT QE curves suggest that between 700-800 there is
some possibility to obtain signal, but the theory may be different from practice.

Has anyone actually attempted this, successfully or otherwise?

Regards,

Dan




On Mon, 25 Jul 2011 23:48:31 -0600, Craig Brideau
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Lenses designed for 2P microscopy might help here.  They tend to have more
>IR correction.  Olympus' XLUMPLFLN or LUMP/IR water dipping lenses are an
>example.  I think most manufacturers have similar lenses.  You will probably
>need to tweak your collection optics however.  The good news is that PMTs
>are fairly tolerant of beam focus differences...  The bad news is that your
>confocal pinhole isn't, and for longer wavelengths you will need to open it
>up anyway.
>
>On a related note, I'm waiting for somebody to use a Cr:Forsterite laser to
>do 2 photon with these NIR dyes.  Cr:Forsterite is similar to Ti:Saph, but
>at 1200-1300nm so it can 2P excite dyes that are further into the NIR.  The
>main limit right now seems to be in the optics.
>
>Craig
>
>
>On Mon, Jul 25, 2011 at 9:38 PM, George McNamara
><[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/**wa?

A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?
A0=confocalmicroscopy>
>> *****
>>
>> Hi Claude,
>>
>> Li-Cor's Odyssey scanner uses APDs. Many FCS confocal microscopes use
APDs
>> - but not necessarily the filter set(s) for fluorophores such as IRDye800
>> (or more clinically, ICG). As for PMTs in the IR: if they have low quantum
>> efficiency but the specimen has practically no autofluorescence (and no
>> non-specific fluorescence of the NIRF), SNR may approach #/0. On the
other
>> hand, infrachromatic [yes, this is a word] correction may be poor (or good),
>> depending on make and model of microscope and especially objective lens -
-
>> not a big deal if you are only looking at one fluorophore. Most back
>> illuminated CCD's have nice QE's in the NIR.
>>
>> Enjoy,
>>
>>
>> George
>> p.s. while looking up infrachromatic on the Internet I came across
>> http://iopscience.iop.org/**0036-
021X/66/3/R04<http://iopscience.iop.org/0036-021X/66/3/R04>   which
reminded me that multiphoton imaging has been around for well over
>> a century. I wonder whether more images were acquired that way or by
cameras
>> and PMTs, APDs etc.  I am not going to trade in my Chameleon or APDs for
a

>> bucket of supersensitizer, but figured worth mentioning at least one
>> alternative has existed for a lng time.
>>
>>
>>
>> On 7/25/2011 10:19 PM, Claude Messier wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/**wa?

A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?
A0=confocalmicroscopy>
>>> *****
>>>
>>> I have been using Alexa 680 and Li-Cor IrDye800 for regular fluorescence
>>> microscopy. I am trying to migrate infra-red dyes to confocal systems
>>> particularly the single-photon variety using spectral detection with a
>>> photomultiplier tube detection. Two problems are obvious the laser source
>>> and the sensitivity of the photomultiplier tube in the infra-red region.
>>> Typical
>>> photomultiplier tubes detector have low recovery when approaching
800nm

>>> so that is a problem. There are infra-red diode lasers close enough to the
>>> excitation peak of IrDye800 to be useable.
>>> My question is: has anyone experimented with these infra-red dyes in
>>> spectral detection system. Any information that would indicate that this
>>> is
>>> possible would be appreciated - most important is whether PMT's can
>>> recover
>>> enough photons at the infra-red end of the sensitivity.
>>> Many thanks
>>>
>>>
>>>
>>
>>
>> --
>>
>>
>> George McNamara, PhD
>> Analytical Imaging Core Facility
>> University of Miami
>>