Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584698.html
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Stanislav,
Coverslip correction refers to imaging a sample in the same refractive index as the coverslip. That is what you are doing in the conventional biological imaging of a fixed specimen. Going back into air effectively negates the correction, and you would be better off with a non-coverslip objective. (But of course you'd have to apply a depth correction).
I cannot offer a solution to imaging through mylar. 40+ years ago I was struggling with this problem and couldn't solve it. (It would have made a really good paper if I could have!)
Guy
Guy Cox, Honorary Associate Professor
School of Medical Sciences
Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006
-----Original Message-----
From: Confocal Microscopy List [mailto:
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Sent: Tuesday, 2 February 2016 3:08 AM
To:
[hidden email]
Subject: Re: reflected light profilometry trrough plastic - mystery bands
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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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