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Kate, that's a good point too. I've had this kind of argument with other people before as well. A TIRF penetration depth of 100 nm does not mean you see only 100 nm deep into the sample (which is how many people interpret the meaning of this number). It simply means the intensity of the evanescent field decays to an intensity of 1/e over that distance. You're right that very bright, high fluorophore concentration objects/vesicles can appear in the image beyond the 100 nm depth. That is to say, it's not so straightforward to translate pixel intensity into object height above the glass coverslip because the intensity is related to the distance from the coverslip AND the fluorophore concentration (and a few other things)... Now, having said all that, there have been several papers in the past that did you show you could determine object heights if you have a good way of capturing images of the same thing at multiple penetration depths. For example:

Olveczky, B.P., N. Periasamy, and A.S. Verkman, Mapping fluorophore distributions in three dimensions by quantitative multiple angle-total internal reflection fluorescence microscopy. Biophysical Journal, 1997. 73(5): p. 2836-2847.

But even there, there are some assumptions made in those kinds of methods. Related to this topic, some people might find of interest this early view paper:

Chiu, C.-L. and E. Gratton, Axial super resolution topography of focal adhesion by confocal microscopy. Microscopy Research and Technique, 2013
http://onlinelibrary.wiley.com/doi/10.1002/jemt.22267/abstract

This method seems much more robust to me and simpler.


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



On 2013-08-21, at 8:22 AM, Kate Luby-Phelps wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> Since the evanescent field decays exponentially as you get away from the refractive index 
> interface, penetration depth is actually somewhat arbitrary in any case. If you have a very 
> high concentration of fluorophore and a sensitive camera you can end up collecting signal 
> deeper than what you calculate from the equation. Direct measurement is best, as already 
> pointed out.
> 
> One other point regarding choice of systems: I would strongly advise choosing a microscope 
> stand with laser autofocus. Otherwise, small temperature fluctuations, etc. will make your 
> timelapse data unusable. Laser autofocus is offered by most if not all the major microscope 
> vendors. I only have experience with Nikon perfect focus, which was a game changer for us 
> after having two systems from two other vendors before hardware autofocus became 
> available.
> 
> Finally, a question for the list - does anyone know if there is a vendor that sells a system 
> with some sort of scrambler to get rid of interference fringes?
> 
> 
> Kate