http://confocal-microscopy-list.275.s1.nabble.com/TIRF-depth-calibration-tp5769934p5770825.html
There is also a simple method in Fiolka et al. Miocroscoc. res Tech 2007 :
inclined coverslip with attached fluorescent beads. I didn't try it myself
> *****
> To join, leave or search the confocal microscopy listserv, go to:
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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:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > *****
> >
> > 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
> >
> > TILL Photonics GmbH
> > Lochhamer Schlag 21
> > D- 82166 Gräfelfing, Germany
> > Phone +49 (0)89 895 662-120
> > Fax +49 (0)89 895 662-101
> > www.till-photonics.com
>