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Yes, Julio, based on a phone call to tech support at Licor, the scanner uses
a laser for excitation and avalanche photodiodes to detect. At the highest
resolution it can do (20 µm) and using the highest sensitivity setting, the
signal from the section was pretty faint. Since the resolution of the 63X
1.4 NA lens we are using is nominally about 0.2 µm, we probably have at
least 100x less signal on the microscope. 2x2 binning didn't help and we
can't afford the loss of spatial resolution with higher binning. I am
doubtful we can do this with a camera and an arc lamp.  We are thinking
about confocal but even if we get enough excitation with 633, it is not
clear that the PMTs will be able to detect out at 800 nm. I haven't tried a
lower mag, high NA lens. I'll have to see what I have or can borrow. 

There doesn't appear to be significant photobleaching on the scanner.
However, it just occurred to me that there might be an autoexposure function
on the scanner that masks photobleaching. I will check. 

There IS photobleaching on the microscope: We took a drop of dye solution
and put it on with no neutral density filters in the light path and it
bleached to invisibility in 5 to 10 secs, which was a surprise and certainly
gave us food for thought. After that we were especially careful to focus by
transmitted light and not open the IRDye 800 shutter until the camera was
already acquiring. Still no signal above background. 

Thanks for all your helpful thoughts.

Kate
On Wed, 9 Jul 2008 15:05:59 -0700, Julio Vazquez <[log in to unmask]> wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>-
>Kate,
>
>You also need to keep in mind that on the Odyssey you are using a  
>laser for optimal excitation, as opposed to a lamp (with low output  
>in the 700 nm range), and possibly also a detector optimized for IR,  
>and that on the Odyssey, even at the highest resolution, your pixel  
>size is several orders of magnitude greater, so you are integrating  
>signal from a huge region compared to a 63x lens.  Also, I guess on  
>the scanner you are integrating the signal from the entire thickness  
>of the section. Maybe I would suggest using a low mag/high NA lens,  
>such as a 10x/0.45 water lens or similar, and 2x2 or 3x3 binning.  
>Using a confocal might also help... if the in-focus signal is quite  
>weak, it may get drowned by the out of focus background (plus the 633  
>nm laser may boost excitation a little).
>
>Are you using fresh slides for the microscope imaging, or slides that  
>were previously scanned on the Odyssey? (just wondering how much  
>bleaching the Odyssey might cause...)
>
>--
>Julio Vazquez,
>Fred Hutchinson Cancer Research Center
>Seattle, WA 98109-1024
>
>
>http://www.fhcrc.org/
>
>
>On Jul 9, 2008, at 12:58 PM, Craig Brideau wrote:
>
>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- 
>> bin/wa?S1=confocal According the the dye's specification sheet it  
>> absorbs at 778 and emits at 806nm. These are fairly close together,  
>> so the filter blocking reflected light may also be blocking your  
>> signal.  You may need a sharper edged dichroic or filter to  
>> separate the excitation light from the signal.
>>
>> Craig
>>
>>
>> On Wed, Jul 9, 2008 at 1:13 PM, Kate Phelps  
>> <[log in to unmask]> wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Does anyone have experience with microscope imaging of very low  
>> IRDye 800 signals in
>> tissue sections? Particularly I am interested in two things:
>>
>> 1) What equipment you are using successfully.
>>
>> 2) Photostability issues.
>>
>> We are using the following setup:
>>
>> Mercury arc lamp, SP-106 filter set from Chroma, Hamamatsu Orca  
>> BT-1024G on a Zeiss
>> Axioimager, 63X 1.4 NA oil immersion lens. Sections are either  
>> paraffin or frozen 10 to
>> 20 µm thick.
>>
>> We have also tried, Xenon arc, halogen cranked up to 12V, 41009  
>> filter set from Chroma.
>> None of these helped much. I have checked for and removed IR  
>> filters at every accessible
>> place in the lightpath. I am sure we are putting a lot of light  
>> into the specimen because
>> you can see it very brightly even though the center wavelength of  
>> the excitation filter is
>> 740 nm. Also, we can detect the autofluorescent signal, just  
>> nothing specific.
>>
>> Scanning the tissue sections on a Licor gel scanner gives a  
>> detectable signal. The control
>> sections are negative on the scanner.
>>
>> Hoping someone can help.
>>
>> Kate Luby-Phelps
>>
>
>