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I very much concur with Jay's statements. I have also noticed that overly confluent cells usually lead to very poor TIRF images, probably for the same reason that he mentioned. In addition to the uncertainty in the penetration depth caused by the sample index of refraction, there are additional scattering within the objective lens that can cause the penetration depth to deviate from single exponential behavior. This has been documented here:

Mattheyses, A.L. and D. Axelrod, Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence. Journal of Biomedical Optics, 2006. 11(1).

and confirmed here:

Oreopoulos, J. and C.M. Yip, Combined scanning probe and total internal reflection fluorescence microscopy. Methods, 2008. 46(1): p. 2-10.

I don't think any of the commercial systems account for this effect as it would vary for each objective lens. Bottom line is, you should calibrate your penetration depth yourself on any commercial system if you really care about its exact value. Most people are usually satisfied with eliminating the out-of-focus light near the cell surface, however, in which case the penetration depth value doesn't matter so much. All of the commercial systems I'm aware of are capable of meeting that requirement.

Sincerely,

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


On 2013-08-20, at 2:32 PM, Unruh, Jay wrote:

> *****
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> 
> Hi all,
> 
> If you intend to work on interfaces other than glass/water, be very skeptical of claims to determine the TIRF field penetration depth.  This is highly refractive index dependent and will depend strongly on cell type/adhesion strength, basement membrane composition, etc...  I have seen scenarios where adherent cells will couple the light out of the coverslip into the solution.  Cell walls will also do funny things with the evanescent wave.
> 
> Nevertheless, the ability to calibrate the incidence angle can be useful for repeatability.  Whether it is worth the cost is another consideration.  Just don't assume that you can set the exact same TIRF angle for every sample type and get the same excitation depth.
> 
> Jay
> 
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Zac Arrac Atelaz
> Sent: Monday, August 19, 2013 11:40 AM
> To: [log in to unmask]
> Subject: Re: Feedback on TIRF systems
> 
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Bryon:
> 
> I have personally worked on the Olympus TIRF, the first versión with everything manual, and the new versión with the automated SIS system, and I have also have the chance to see the http://www.tirftechnologies.com/ system insert working with an Olympus IX81 microscope.
> 
> The manual TIRF from Olympus was a single user system as many manual adjustments were needed. Here is a great BUT, the new automated system is really impressive, you can adjust the depth from which you are having signal coming out. So the electromagnetic space adjacent to your coverglass is greatly controlled. All the laser alignment is made only once by the installation engineers and basically remains like that for life, they work now with led lasers, so maintenance would not be an issue, and expected life for leds is quite long.
> 
> Tirf tech have a very small system which is interesting, you might want to take a look to that one also.
> 
> I have not Heard from other TIRF systems 
> 
> Best regards and happiness
> 
> Gabriel OH
> 
>> Date: Fri, 16 Aug 2013 11:49:38 -0500
>> From: [log in to unmask]
>> Subject: Feedback on TIRF systems
>> To: [log in to unmask]
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Dear All,
>> 
>> 
>> 
>> We are currently looking at TIRF/widefield fluorescence microscopy 
>> systems from Olympus, Zeiss and DeltaVision and are seeking comments 
>> from users of these systems. We would greatly appreciate any feedback 
>> that users could provide. Since this is not directly a confocal 
>> microscopy question, I would be happy to receive comments directly 
>> ([log in to unmask]).
>> 
>> 
>> 
>> Thanks so much,
>> 
>> 
>> 
>> Bryon
>> 
>> 
>> 
>> *******************************
>> 
>> 
>> 
>> Bryon Grove, Ph.D.
>> 
>> Associate Professor
>> 
>> Department of Basic Sciences
>> 
>> Director, Edward C Carlson Imaging and Image Analysis Core Facility
>> 
>> UND School of Medicine and Health Sciences
>> 
>> 501 N Columbia Rd Stop 9037
>> 
>> Grand Forks, ND, 58202-9037
>> 
>> Phone: 701-777-2579
>> 
>> Fax: 701-777-2477
>