Posted by
Stanislav Vitha-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584695.html
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Guy,
I agree the setup has problems. Yes, the objective is coverslip corrected.
Thanks for the reference to your Micron paper.
I think the multiple reflections are a good possibility, or the strange effects
of birefringence.
regarding the birefringence, are there any good tricks ti minimize the
problem in this setup? Like putting a 1/4 lambda plate somewhere in the
lightpath?
I do not quite understand your comment regarding spherical aberration
when imaging with dry objective into an air cavity. I thought we have a
perferct RI match there (air objective, air in the specimen) so no SA would
be expected providing the coverglass thickness is correct.
I also tried an objective with coverglass adjustment (0 to 2 mm range) and
optimized the setting for maximum signal (= minimum SA) for the
reflection from the first surface of the channel. The results were similar.
I will test my 10x/0.4 objective and see what I get, but I will probably end
up filling the channel with fluorescencent due solution and measure it this
way. It will be interesting to compare the two methods.
Stan Vitha
Microscopy and Imaging Center
Texas A&M University
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On Sat, 30 Jan 2016 01:08:23 +0000, Guy Cox wrote:
This is truly an optically horrible setup. I'm assuming that you are using a
coverslip-corrected objective and therefore that your top 170µm of mylar
won't be too far out. But this is not designed for then imaging into an air
cavity, so you will have major problems with spherical aberration. To make
matters worse, mylar is birefringent so your polarized beam of laser light
will be split into two beams, each perceiving a different refractive index and
therefore aberrated differently. For an example of how SA can screw up
depth measurements see G.C. Cox and C.J.R. Sheppard, 2001.
Measurement of thin coatings in the confocal microscope. Micron 32, 701-
705.
There is another possible explanation, and that is that you are seeing
multiple passes through the cavity. So at 20µm you are seeing the true
reflection, then 40µm down you are not seeing light from there at all, but
light that has been reflected twice in the cavity, etc. This would explain
why all your peaks are multiples of the expected cavity depth.
Paradoxically, I'd suggest using a worse optical system, such as a 10x NA
0.3 objective. This will reduce the effects of SA. Your reflection lines will
now be much broader and your scans will look terrible BUT the true surface
will still be at the brightest point (neglecting any residual SA). So a line
profile across your scan will show you the true depth.
Guy
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