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Hi Kyle,
what works very well in my group is a dye sandwich between coverslip and coverslide. Use a small amount (~1ul) of not too diluted dye solution on a cleaned coverslide, put the clean coverslip on top and move it around until you cannot move it any more. Then you have a very thin dye layer. Seal it at the sides with nail polish, then the sample lasts longer.
You won’t have any out-of-focus light in this case. Don’t use too high powers, otherwise you start bleaching in the center and the dyes cannot replenish quickly enough.
Good luck,
Jonas Ries
> On 03 Jun 2015, at 17:02, John Oreopoulos <[log in to unmask]> 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.
> *****
>
> Kyle,
>
> Please see this link:
>
> http://spectral.ca/Downloads?f=2745809748.pdf
>
> The protocol described there is a modification of that originally presented by Michael Model (Kent State University) in the following publications:
>
> Model, M.A. and J.L. Blank, Intensity calibration of a laser scanning confocal microscope based on concentrated dyes. Analytical and Quantitative Cytology and Histology, 2006. 28(5): p. 253-261.
>
> Since writing my version of the protocol, another dye has been identified that is compatible with 405 nm excitation. The dye is called pryanine and is available from Invitrogen/Life Technologies and Sigma Aldrich (no commercial interest).
>
> The protocol was developed to measure illumination/emission uniformity of confocal microscopes, but it does work for widefield fluorescence as well. The main issue is that you will see the out of focus light associated with the thin layer of fluorescence near the coverslip. The confocal microscope rejects this light.
>
> Sincerely,
>
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research Inc.
> A Division of Andor Technology
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
>
>
>
>
> On 2015-06-03, at 10:42 AM, Kyle Douglass 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 everyone,
>>
>> I would like to check the illumination uniformity on a widefield laser epi-illumination fluorescence microscope. I am familiar with a few older publications that provide protocols for producing uniform rhodamine/fluoresceine test slides for this purpose, but a quick survey of their methods and across the internet led me to believe that few people have tried these protocols with a 100x/1.45 NA objective lens.
>>
>> Is anyone willing to share a proven protocol for preparing fluorescent dye-based test slides for measuring illumination uniformity in a widefield microscope with a 100x/1.45 NA objective? I am concerned that the very small axial extent of the focal volume of a high magnification objective would make this measurement tricky because of out-of-focus fluorescence corrupting the measurement.
>>
>> I can see two strategies for doing this, but I am not sure which one would be best:
>> 1) Use such a large concentration of dye that the laser light is absorbed before it propagates further than the axial extent of the focal volume;
>> 2) Make the dye layer so thin that it's roughly the same size as the axial extent focal volume
>>
>> Any feedback would be appreciated. Thanks!
>>
>> Kyle
>>
>> --
>> Kyle M. Douglass, PhD
>> Post-doctoral researcher
>> The Laboratory of Experimental Biophysics
>> EPFL, Lausanne, Switzerland
>> http://kmdouglass.github.io
>> http://leb.epfl.ch
---
Dr. Jonas Ries
- group leader -
Cell Biology and Biophysics Unit
European Molecular Biology Laboratory
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