Posted by
Stanislav Vitha-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584699.html
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Guy,
I must be a bit dense, but your explanation does not make sense for e.g.
water immersion objectives.
When coverslip correction on these lenses is applied, as long as the
refractive index of the sample (water) is exactly the same as the design
immersion medium (water), spherical aberration is corrected throughout
the imaging depth (whatever the working distance permits).
So essentially, a coverslip of proper thickness (corresponding to the
settings of the correction collar, or to the fixed design thickness of the
objective) can be positioned anywhere between the objective front element
and the specimen.
I believe the same concept applies to dry lenses.
I have seen a paper that I need to dig out, where this was explained (with
math) and experimentally shown for dry lenses.
So if you have a dry objective designed for 0.17mm coverslip and need to
look at the first surface of a naked specimen, you could attach the coverslip
to the front of the objective and achieve SA correction. I can confirm that
this works when I need to do reflected brightfield or reflected DIC of
surfaces with 20x/0.5 and 40x/0.75 lenses on my old Zeiss Axiophot. A
careful smear of silicone grease on the metal surrounding the (concave)
front lens will hold the coverslip nicely.
Bit I am getting a bit off topic.
I am about to fill the microchannel with rhodamine dye solution and
measure the depth by fluorescence.
I will report back how it went.
Stan Vitha
On Tue, 2 Feb 2016 11:53:35 +0000, Guy Cox
<
[hidden email]> wrote:
>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:
[hidden email]] On Behalf Of Stanislav
Vitha
>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
>
>
>
>=====
>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
>===========