Re: TIRF depth calibration

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-depth-calibration-tp5769934p5770753.html

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Sebastian, I remember reading a publication from about ten years ago that talked about mounting a pencil in a piezoelectric micro-manipulator and sticking a fluorescent bead on the end of the pencil tip. This was very similar to the AFM method. Is it the same thing?

Graham, there is a fairly detailed discussion on this topic from a few years ago on the archive that talks about a few other ways to measure the actual TIRF penetration depth (as opposed to calculating it based on an assumed refractive index and crudely measured incident angle):

http://lists.umn.edu/cgi-bin/wa?A3=ind0702&L=CONFOCALMICROSCOPY&E=quoted-printable&P=827770&B=--Apple-Mail-181-588547976&T=text%2Fhtml;%20charset=ISO-8859-1

I've tried the AFM method as well - it works, but my main complaint with this and some of the other protocols is that they require complicated and expensive equipment, and can be difficult to get right. The method involving fluorescent microtubles by Jorg Enderlein's group is a fairly new one that is elegantly simple, but again requires you to have access to some fairly special reagents that might not be found in every lab. A few months ago I came across yet another older method that had evaded my previous searches on the topic. This one is similar to the others that involve imaging fluorescent microbeads, but I like this because all it requires is a microscope with a motorized drive on the z-axis:

Steyer, J.A. and W. Almers, Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophysical Journal, 1999. 76(4): p. 2262-2271.

John Oreopoulos
Research Assistant
Spectral Applied Research
9078 Leslie Street, Unit 11
Richmond Hill
Ontario, Canada


On 2010-11-24, at 6:54 AM, Sebastian Rhode wrote:

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> Hi Graham,
>
> one method which definitly works (I tried it out by myself) is the use of an
> combined TIRF-AFM setup. You just have to couple fluorescent beads to the
> tip of your AFM an record pictures while approaching/or moving away the
> coverslip surface. Unfortunately an AFM is really expensive.
>
> So I found some other methods, which might work as well --> see
> TIRF_Introduction.pdf, which I send to you directly (LIST server does not
> accepted this pdf-file).
>
> One methodes uses an objective piezo-drive and a pencil and the second one
> stained beads or a stained solution with intransparent beads.
>
> I case of questions, feel free to contact me directly.
>
> Cheers,
> Sebastian
>
>
> Dr. Sebastian Rhode
> Project Manager
> Research & Development
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