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Hi Renato,

Yes, reflection confocal microscopy works great for this. You may need 
to disable a reflection suppression option to get maximum signal 
(hopefully a readily visible checkbox in the GUI - for example, the 
Leica LAS AF option is harder to find than Zeiss ZEN option). Evaluate 
each of your laser lines for best contrast and least noise. Evaluate 
different combinations of laser power and detector gain (ex. high laser 
power, low gain, assuming the laser line is not noisy). Probably 
simplest to leave the pinhole at 1.0 Airy units, though smaller might 
result in even better resolution. Hopefully they have controls - there 
are usually some background grains that need to be counted. Grains 
distributed in Z may or may not be due to 35S. Silver grain size may not 
be proportional to the amount of 35S in a given volume - and grain count 
may be linear over a limited range (though may not be practical to 
evaluate this).

If all the laser lines perform well, you can choose the channel that 
gives the best counterstain image in the transmitted light detector. 
Note that some counterstains - notably eosin - are also fluorescent. If 
you / your colleague want a color transmitted light image, do this with 
3 laser lines, such as 458 (Ar), 561 (DPSS), 633 (HeNe). The laser lines 
do not need to be perfectly "RGB", just close. To speed up imaging, you 
could just acquire two color channels and use one twice, such as 633 nm 
for R, and 488 nm for G and B. If the silver grains also show up in the 
transmitted light images, not a big deal: you have them in high contrast 
in the reflection confocal image. There was a recent article 
(Biotechniques?) whose whole point was that they were able to use LacZ 
beta galactosidase (BCIP  or NBT/BCIP) product fluorescence confocal 
images to identify what black voids were product and what were 
irrelevant junk from the specimen.

For image acquisition, more frame averaging is better - since silver 
grains will not photobleach, you can average as much as you want (if 
this is a core facility, you could maximize revenue by using maximum 
averaging).

Hopefully your confocal microscope software include deconvolution (if 
you have Leica LAS AF try the different settings). If not, the Bruce and 
Butte NVidia CUDA based GPU deconvolution software
http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375 
<http://www.opticsinfobase.org/oe/fulltext.cfm?uri=oe-21-4-4766&id=249375>
is fast and no charge for academic license - just need to complete and 
send to Manish the end user license agreement
http://tcell.stanford.edu/software.html
If you use it, do not just accept the -1 (automatic) default for number 
of iterations - evaluate different settings. I typically use 100 
iterations on a Quadro 2000 card (about 6 seconds for 512x512x32 
planes). I just ordered the new GeForce GTX 780 Ti (3 Gb card, about 
$700), which I expect will enable me to deconvolve full FLASH4.0 
(2048x2048 pixels) fields of view. In addition to X and Y being a power 
of two (beneficial for FFTs), Manish and I recently exchanged emails 
where they reminded me that Z should also be a power of 2 for both speed 
and GPU ram efficiency (probably true of all deconvolution software).


Sincerely,

George


On 11/25/2013 4:29 AM, Renato Mortara wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear friends,
>
>
>
> A colleague is trying to image mouse brain sections that were radioactively
> labeled (I guess 35S-Methionine) then, immersed in a photographic emulsion
> to develop particular sites.
>
>
>
> The sections were then counter stained with toluidine or similar (bluish)
> histological dye. The final result ('bright spots, easily seen under a 20x
> objective) can be visualized under dark field microscopy.
>
>
>
> I wonder if anyone has been able to image through some kind of reflection
> mode under confocal microscopy.
>
>
>
> Thanks !
>
>
>
> Renato Mortara
>
>
>
> Dr. Renato Arruda Mortara
>
> Parasitology Division
>
> Escola Paulista de Medicina - UNIFESP
>
> Rua Botucatu,  862 6th floor
>
> 04023-062
>
> São Paulo SP Brazil
>
> Phone: 55 11 55798306
>
> www.ecb.epm.br/~ramortara
>
>    


-- 



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/