Posted by
mcammer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/TIRF-depth-calibration-tp5769934p5770973.html
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Related to the axial resolution issue (which I'll briefly address at the end), I have a question regarding the uniformity of excitation over the TIRF field.
At the end of
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-8859-1there is a reference to a solution to non-uniformity of excitation across the TIRF field due to the coherent light source. The solution is to spin a wedge in the light source and try to have multiple spins per exposure time.
I recall back in the early 1990s reading a paper on Nomarski optics to image microtubule dynamics in vitro (maybe M Sheetz or RD Vale?) which used a green laser for illumination and spinning diffusion filter in the light path to remove coherence and, thus, speckle.
Does anybody have such a modification to the light path and would you please share your results?
On the axial resolution issue, about six years ago to estimate depth penetration of the TIRF field in the simple case of a glass, thin protein coating, and PBS interface with the Olympus TIRF system of the time, we stuck 4 um Tetraspeck beads to a 1.5 coverslip with polylysine. As we adjusted the micrometer the beads appeared smaller and larger. Using simple trig, we estimated the depth of the field. Of course, this was far from perfect since the beads were big and tend to squish down where they stick, but we got a rough idea.
-Michael
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From: Confocal Microscopy List [mailto:
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Sent: Wednesday, November 24, 2010 10:33 AM
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Subject: Re: TIRF depth calibration
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