Advice on citations for homogenized fluorescence illumination

[Ricardo Henriques-2]

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Hello everyone,

I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.

Thank you in advanced.
Best regards,
Ricardo Henriques

Ricardo Henriques
Instituto de Medicina Molecular (Lisbon, Portugal).
For contact information see: https://sites.google.com/site/paxcalpt/

[John Oreopoulos]

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Partial commercial response:

Hi Ricardo,

There are a number of fairly recent research articles which come to mind. I have collected a number of references below which you may find helpful.
In a related commercial response, I would like to add that the company that I work for, Spectral Applied research, developed a laser-based illumination technology named Borealis (patent-pending) for spinning disk confocal microscopes a few years ago. Although not the original intent of the technology, one of the benefits of Borealis is that it provides, to the best of our knowledge, the flattest and most homogenous illumination profile for any laser-based microscope illumination system. Since I joined the company a year ago, we've spent a great deal of time trying to quantitatively prove this in the R&D lab here at Spectral. Mike Model's concentrated dye solutions were particularly helpful in this regard (see references below). We measure illumination uniformity as the percent rolloff from the peak intensity in the middle to the lowest intensity at the ends of a line profile drawn across an image diagonal (as recommended in Pawley's confocal handbook). Spinning disk confocal systems out of the box are notoriously bad for uniformity and in some cases we measured a %rolloff as high as 60% in the red portions of the visible spectrum. With Borealis, we can bring this down 5% or less. When other optical system non-uniformities are included (camera lens field curvature and vignetting for example), this value is still on the order of only 8%. We are currently compiling all of this data into a publishable form. In addition, Borealis can be easily adapted for regular wide-field epfluorescence microscopes as well. We'll be demoing the wide-field version of Borealis at the upcoming Neuroscience and Cell Biology annual meetings.

The references I found useful over the years regarding microscope illumination uniformity are:

1. Waters, J.C., Accuracy and precision in quantitative fluorescence microscopy. Journal of Cell Biology, 2009. 185(7): p. 1135-1148.
2. Murray, J.M., et al., Evaluating performance in three-dimensional fluorescence microscopy. Journal of Microscopy-Oxford, 2007. 228(3): p. 390-405.
3. Wolf, D.E., C. Samarasekera, and J.R. Swedlow, Quantitative analysis of digital microscope images, in Digital Microscopy, 3rd Edition. 2007, Elsevier Academic Press Inc: San Diego. p. 365-396.

Wolf's chapter in Digital Microscopy talks about why there is a need for flat illumination in the context of an quantitative measurements with a fluorescence microscope, especially in cases where comparisons are made across different spectral channels (FRET, calcium imaging, etc.).

Next:

4. Zucker, R.M. and O.T. Price, Practical confocal microscopy and the evaluation of system performance. Methods-a Companion to Methods in Enzymology, 1999. 18(4): p. 447-458.
5. Zucker, R.M. and O. Price, Evaluation of confocal microscopy system performance. Cytometry, 2001. 44(4): p. 273-294.

Zucker talks about some alternative methods to assess microscope illumination Uniformity. You might also want to take a look at these references for some more ideas and methods:

6. Zwier, J.M., et al., Image calibration in fluorescence microscopy. Journal of Microscopy-Oxford, 2004. 216: p. 15-24.
7. Brakenhoff, G.J., et al., Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts. Journal of Microscopy-Oxford, 2005. 219: p. 122-132.
8. Zwier, J.M., et al., Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures. Journal of Microscopy-Oxford, 2008. 231(1): p. 59-69.

9. Model, M.A. and J.K. Burkhardt, A standard for calibration and shading correction of a fluorescence microscope. Cytometry, 2001. 44(4): p. 309-316.
10. Model, M.A., Intensity Calibration and Shading Correction for Fluorescence Microscopes. Current Protocols in Cytometry. 2006: John Wiley & Sons, Inc.
11. 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.
12. Model, M.A. and J.L. Blank, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope. Journal of Microscopy-Oxford, 2008. 229(1): p.12-16.

Mike Model's "concentrated fluorescent dye" solutions for measuring system uniformity are very easy to make and have some other useful properties, including intensity calibration. Finally, there are two publications from this year, one that actually tried to catalog the non-uniformities across different microscope systems (mainly confocal) during an international standardization study:

13. Stack, R.F., et al., Quality Assurance Testing for Modern Optical Imaging Systems. Microscopy and Microanalysis, 2011. 17(4): p. 598-606.
14. Michálek, J., M. Čapek, and L. Kubínová, Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy. Microscopy Research and Technique, 2011. 74(9): p. 831-838.

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2011-10-03, at 8:12 AM, Ricardo Henriques wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello everyone,
>
> I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.
>
> Thank you in advanced.
> Best regards,
> Ricardo Henriques
>
> Ricardo Henriques
> Instituto de Medicina Molecular (Lisbon, Portugal).
> For contact information see: https://sites.google.com/site/paxcalpt/

[Chen, De (NIH/NCI) [C]]

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Hi Ricardo:

The non-uniform illuminaiton can be corrected post-acquisition which can be done by the polynomial fitting to each row of the image intensity profile and followed by a subtraction.


________________________________________
From: Ricardo Henriques [[hidden email]]
Sent: Monday, October 03, 2011 8:12 AM
To: [hidden email]
Subject: Advice on citations for homogenized fluorescence illumination

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To join, leave or search the confocal microscopy listserv, go to:
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Hello everyone,

I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.

Thank you in advanced.
Best regards,
Ricardo Henriques

Ricardo Henriques
Instituto de Medicina Molecular (Lisbon, Portugal).
For contact information see: https://sites.google.com/site/paxcalpt/

[John Oreopoulos]

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There's one other reference I remembered written back in 2003 that summarizes quite nicely the state of the art at that time for regular, wide-field epifluorescence microscopy:

1.  Dimas, C.F., J.J. Kuta, and M. Hubert, An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy, in Applications of Photonic Technology 6 - Closing the Gap between Theory, Development, and Application, R.A. Lessard and G.A. Lampropoulos, Editors. 2003, Spie-Int Soc Optical Engineering: Bellingham. p. 558-567.

And in the context of TIRF microscopy, we've discussed this topic before on the confocal list server. See here:

http://lists.umn.edu/cgi-bin/wa?A2=ind1011&L=CONFOCALMICROSCOPY&D=0&P=18312

The relevant articles are:

2. Fiolka, R., et al., Even illumination in total internal reflection fluorescence microscopy using laser light. Microscopy Research and Technique, 2008. 71(1): p. 45-50.
3. Mattheyses, A.L., K. Shaw, and D. Axelrod, Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle. Microscopy Research and Technique, 2006. 69(8): p. 642-647.
4. van't Hoff, M., V. de Sars, and M. Oheim, A programmable light engine for quantitative single molecule TIRF and HILO imaging. Optics Express, 2008. 16(22): p. 18495-18504.

John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca

On 2011-10-03, at 10:52 AM, Chen, De (NIH/NCI) [C] wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Ricardo:
>
> The non-uniform illuminaiton can be corrected post-acquisition which can be done by the polynomial fitting to each row of the image intensity profile and followed by a subtraction.
>
>
> ________________________________________
> From: Ricardo Henriques [[hidden email]]
> Sent: Monday, October 03, 2011 8:12 AM
> To: [hidden email]
> Subject: Advice on citations for homogenized fluorescence illumination
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello everyone,
>
> I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.
>
> Thank you in advanced.
> Best regards,
> Ricardo Henriques
>
> Ricardo Henriques
> Instituto de Medicina Molecular (Lisbon, Portugal).
> For contact information see: https://sites.google.com/site/paxcalpt/

[Ricardo Henriques-2]

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Hi John,

This is extremely useful. I'm grabbing now the literature and will check it out.
I'll write you an email outside of the list.
Thank you :),
R

Ricardo Henriques
Instituto de Medicina Molecular (Lisbon, Portugal).
For contact information see: https://sites.google.com/site/paxcalpt/

On Oct 3, 2011, at 6:48 PM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There's one other reference I remembered written back in 2003 that summarizes quite nicely the state of the art at that time for regular, wide-field epifluorescence microscopy:
>
> 1.  Dimas, C.F., J.J. Kuta, and M. Hubert, An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy, in Applications of Photonic Technology 6 - Closing the Gap between Theory, Development, and Application, R.A. Lessard and G.A. Lampropoulos, Editors. 2003, Spie-Int Soc Optical Engineering: Bellingham. p. 558-567.
>
> And in the context of TIRF microscopy, we've discussed this topic before on the confocal list server. See here:
>
> http://lists.umn.edu/cgi-bin/wa?A2=ind1011&L=CONFOCALMICROSCOPY&D=0&P=18312
>
> The relevant articles are:
>
> 2. Fiolka, R., et al., Even illumination in total internal reflection fluorescence microscopy using laser light. Microscopy Research and Technique, 2008. 71(1): p. 45-50.
> 3. Mattheyses, A.L., K. Shaw, and D. Axelrod, Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle. Microscopy Research and Technique, 2006. 69(8): p. 642-647.
> 4. van't Hoff, M., V. de Sars, and M. Oheim, A programmable light engine for quantitative single molecule TIRF and HILO imaging. Optics Express, 2008. 16(22): p. 18495-18504.
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
> On 2011-10-03, at 10:52 AM, Chen, De (NIH/NCI) [C] wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Ricardo:
>>
>> The non-uniform illuminaiton can be corrected post-acquisition which can be done by the polynomial fitting to each row of the image intensity profile and followed by a subtraction.
>>
>>
>> ________________________________________
>> From: Ricardo Henriques [[hidden email]]
>> Sent: Monday, October 03, 2011 8:12 AM
>> To: [hidden email]
>> Subject: Advice on citations for homogenized fluorescence illumination
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello everyone,
>>
>> I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.
>>
>> Thank you in advanced.
>> Best regards,
>> Ricardo Henriques
>>
>> Ricardo Henriques
>> Instituto de Medicina Molecular (Lisbon, Portugal).
>> For contact information see: https://sites.google.com/site/paxcalpt/

[John Oreopoulos]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Keep in mind with the TIRF illumination schemes that spin the focused laser beam in the back-focal plane one thing: They do an excellent job of removing interference fringes caused by dust particles in the optical train and fringes caused by refractive index mismatches near the glass-water interface, but they don't remove the overall Gaussian profile of the illumination at the sample plane.

White light TIRF microscopes don't really suffer this issue, but they have other problems to deal with. See this early paper by Andrea Stout and Dan Axelrod:

1. Stout, A.L. and D. Axelrod, Evanescent Field Excitation of Fluorescence by Epi-Illumination Microscopy. Applied Optics, 1989. 28(24): p. 5237-5242.

John Oreopoulos


On 2011-10-03, at 1:12 PM, Ricardo Henriques wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi John,
>
> This is extremely useful. I'm grabbing now the literature and will check it out.
> I'll write you an email outside of the list.
> Thank you :),
> R
>
> Ricardo Henriques
> Instituto de Medicina Molecular (Lisbon, Portugal).
> For contact information see: https://sites.google.com/site/paxcalpt/
>
> On Oct 3, 2011, at 6:48 PM, John Oreopoulos wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> There's one other reference I remembered written back in 2003 that summarizes quite nicely the state of the art at that time for regular, wide-field epifluorescence microscopy:
>>
>> 1.  Dimas, C.F., J.J. Kuta, and M. Hubert, An enhanced method of obtaining uniform excitation radiation for fluorescence microscopy, in Applications of Photonic Technology 6 - Closing the Gap between Theory, Development, and Application, R.A. Lessard and G.A. Lampropoulos, Editors. 2003, Spie-Int Soc Optical Engineering: Bellingham. p. 558-567.
>>
>> And in the context of TIRF microscopy, we've discussed this topic before on the confocal list server. See here:
>>
>> http://lists.umn.edu/cgi-bin/wa?A2=ind1011&L=CONFOCALMICROSCOPY&D=0&P=18312
>>
>> The relevant articles are:
>>
>> 2. Fiolka, R., et al., Even illumination in total internal reflection fluorescence microscopy using laser light. Microscopy Research and Technique, 2008. 71(1): p. 45-50.
>> 3. Mattheyses, A.L., K. Shaw, and D. Axelrod, Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle. Microscopy Research and Technique, 2006. 69(8): p. 642-647.
>> 4. van't Hoff, M., V. de Sars, and M. Oheim, A programmable light engine for quantitative single molecule TIRF and HILO imaging. Optics Express, 2008. 16(22): p. 18495-18504.
>>
>> John Oreopoulos
>> Research Assistant
>> Spectral Applied Research
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>>
>> On 2011-10-03, at 10:52 AM, Chen, De (NIH/NCI) [C] wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi Ricardo:
>>>
>>> The non-uniform illuminaiton can be corrected post-acquisition which can be done by the polynomial fitting to each row of the image intensity profile and followed by a subtraction.
>>>
>>>
>>> ________________________________________
>>> From: Ricardo Henriques [[hidden email]]
>>> Sent: Monday, October 03, 2011 8:12 AM
>>> To: [hidden email]
>>> Subject: Advice on citations for homogenized fluorescence illumination
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hello everyone,
>>>
>>> I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.
>>>
>>> Thank you in advanced.
>>> Best regards,
>>> Ricardo Henriques
>>>
>>> Ricardo Henriques
>>> Instituto de Medicina Molecular (Lisbon, Portugal).
>>> For contact information see: https://sites.google.com/site/paxcalpt/

[Xuejun Sun]

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Hello, everyone,

One of the two fans on our Coherent Verdi-5 controller died and need to be
replaced. This is the fan which is on the heat sink of the LBO crystal
block.

Anyone has done this in house instead of by a Coherent service engineer? I
am able to get the fan and wondering if we should replace it by ourselves
(which will be quicker and cheaper).

Thanks in advance,

Xuejun


Xuejun SUN, Ph.D.
Dept. Exp. Oncology
Cross Cancer Institute
11560  University Ave.
Edmonton Alberta, T6G1Z2

Phone  [phone] (office)
           [phone]/8458 (lab)
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[Monique Vasseur]

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Hi Chen

What do you mean by polynomial fitting? What is your formula?  Thanks in advance.

Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Chen, De (NIH/NCI) [C]
Envoyé : 3 octobre 2011 10:53
À : [hidden email]
Objet : Re: Advice on citations for homogenized fluorescence illumination

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Hi Ricardo:

The non-uniform illuminaiton can be corrected post-acquisition which can be done by the polynomial fitting to each row of the image intensity profile and followed by a subtraction.


________________________________________
From: Ricardo Henriques [[hidden email]]
Sent: Monday, October 03, 2011 8:12 AM
To: [hidden email]
Subject: Advice on citations for homogenized fluorescence illumination

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

I'm preparing a small manuscript that in part covers the concept that most fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous illumination of the field of view - typically gaussian-shaped, less intensity on the image extremities. I was wondering if you could advise me on what you consider the most interesting publications tackling or approaching this subject.

Thank you in advanced.
Best regards,
Ricardo Henriques

Ricardo Henriques
Instituto de Medicina Molecular (Lisbon, Portugal).
For contact information see: https://sites.google.com/site/paxcalpt/

[tineke vendrig]

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For a homogenous illumination you can re-install the lasers and perform
a Köhler illumination again for the best illumination. Köhler illumination
acts to generate an extremely even illumination of the sample and ensures
that an image of the illumination source is not visible in the
resulting image. Köhler illumination is the predominant technique for sample
illumination in modern scientific light microscopy although it requires
additional optics which less expensive and simpler light microscopes.
Good luck!

Tineke Vendrig, ing
technical engeneer optical microscopy
Delft University of Technology
Bionano Science
Kavli Institute of Nanoscience
Lorentzweg 1
2628LJ Delft
room F185
Tel: [phone]
Fax:[phone]
2011/10/3 Ricardo Henriques <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello everyone,
>
> I'm preparing a small manuscript that in part covers the concept that most
> fluorescence microscopes (SD, WF, TIRF, ...) have a non-homogenous
> illumination of the field of view - typically gaussian-shaped, less
> intensity on the image extremities. I was wondering if you could advise me
> on what you consider the most interesting publications tackling or
> approaching this subject.
>
> Thank you in advanced.
> Best regards,
> Ricardo Henriques
>
> Ricardo Henriques
> Instituto de Medicina Molecular (Lisbon, Portugal).
> For contact information see: https://sites.google.com/site/paxcalpt/
>