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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 [[log in to unmask]]
>>> Sent: Monday, October 03, 2011 8:12 AM
>>> To: [log in to unmask]
>>> 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/