Silver enhanced autoradiography / dark field under confocal ?

[Renato Mortara]

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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: [phone]

www.ecb.epm.br/~ramortara

[Guy Cox-2]

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Renato,

          I have (years ago) done silver-enhanced gold immunolabelling in reflection mode confocal microscopy.  It stands out brilliantly.  Labelling that is invisible in transmitted light is very strong in confocal.  It gives you an extra channel over and above what you are imaging in fluorescence, with no cross-talk.  I also discovered that the images are so 'different' that referees don't like them!

                                                                                Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Monday, 25 November 2013 9:30 PM
To: [hidden email]
Subject: Silver enhanced autoradiography / dark field under confocal ?

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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: [phone]

www.ecb.epm.br/~ramortara

[George McNamara]

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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: [phone]
>
> 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/

[mcammer]

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Back in the early 1990s we used reflectance confocal to image in situ.  Also, enhanced immunogold and lead ATPase.  No one has asked for it recently, but even after a decade it must still work.

===========================================================================
Michael Cammer, Microscopy Core & Dustin Lab , Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   Lab: 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://www.med.nyu.edu/skirball-lab/dustinlab/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Monday, November 25, 2013 5:30 AM
To: [hidden email]
Subject: Silver enhanced autoradiography / dark field under confocal ?

*****
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: [phone]

www.ecb.epm.br/~ramortara

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[Jacqueline Ross]

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Hi Renato,

I also imaged the same type of specimens - bovine cartilage explants, silver grains in photographic emulsion using reflectance confocal on a Leica TCS 4D confocal microscope. We published a couple of papers using this technique combined with immunostaining for type VI collagen together with biochemical analysis of the various tissue fractions.

It worked brilliantly for assessing localisation.

I can send PDFs of the papers offline if your colleague is interested.

Kind regards,

Jacqui


Jacqueline Ross
Biomedical Imaging Microscopist
Biomedical Imaging Research Unit 
School of Medical Sciences 
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142, NEW ZEALAND

Tel: [phone]
Fax: [phone]

http://www.fmhs.auckland.ac.nz/sms/biru/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
Sent: Monday, 25 November 2013 11:30 p.m.
To: [hidden email]
Subject: Silver enhanced autoradiography / dark field under confocal ?

*****
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: [phone]

www.ecb.epm.br/~ramortara

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Paddock and Eliceiri, chapter 2 in Paddock 2013, page 33, figure 15
illustrates the benefit of reflection confocal microscopy of silver
grains compared to transmitted light darkfield or transmitted light
brightfield.
http://www.amazon.com/Confocal-Microscopy-Methods-Protocols-Molecular/dp/1588293513

Figure was previously published in Paddock 2002 Biotechnoqies, figure 3,
http://www.biotechniques.com/multimedia/archive/00014/paddock322_14678a.pdf


On 11/26/2013 10:54 PM, Jacqui Ross wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Renato,
>
> I also imaged the same type of specimens - bovine cartilage explants, silver grains in photographic emulsion using reflectance confocal on a Leica TCS 4D confocal microscope. We published a couple of papers using this technique combined with immunostaining for type VI collagen together with biochemical analysis of the various tissue fractions.
>
> It worked brilliantly for assessing localisation.
>
> I can send PDFs of the papers offline if your colleague is interested.
>
> Kind regards,
>
> Jacqui
>
>
> Jacqueline Ross
> Biomedical Imaging Microscopist
> Biomedical Imaging Research Unit
> School of Medical Sciences
> Faculty of Medical&  Health Sciences
> The University of Auckland
> Private Bag 92019
> Auckland 1142, NEW ZEALAND
>
> Tel: [phone]
> Fax: [phone]
>
> http://www.fmhs.auckland.ac.nz/sms/biru/
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato Mortara
> Sent: Monday, 25 November 2013 11:30 p.m.
> To: [hidden email]
> Subject: Silver enhanced autoradiography / dark field under confocal ?
>
> *****
> 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: [phone]
>
> 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/