Illumination field uniformity at very high magnifications

[Kyle Michael Douglass]

*****
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

[Kevin Ryan]

I would recommend a _thick_ dye slide - high magnification fluorescent objectives aren't guaranteed to be completely planar, and you don't want planarity of focus or an angled stage to confuse the illumination measurement. It won't make much of a difference for wide-field, much more so for confocal point-scanning with the confocality of illumination and single pixel dwell times, but still...

The simplest illumination test sample I've used was just an O-ring gasket on a slide, pipetted with a slight excess of dye (convex raised meniscus), and with a cover slip gently dropped on - the surface tension of the dye will pull it down forcing out any excess, usually without leaving any bubbles, giving you a flat test sample. It's simple enough to run on a daily basis.

Kevin Ryan
Media Cybernetics, Inc.



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kyle Douglass
Sent: Wednesday, June 03, 2015 10:42 AM
To: [hidden email]
Subject: Illumination field uniformity at very high magnifications

*****
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

[John Oreopoulos]

*****
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

[Kyle Michael Douglass]

*****
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.
*****

Hi John and Kevin,
Thank you for your responses. I'll give these protocols a try and see
how they work.

Kyle

On 06/03/2015 05:02 PM, John Oreopoulos 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

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Jonas Ries]

*****
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.
*****

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 <[hidden email]> 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