Posted by
Guy Cox-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584689.html
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
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 Craig Brideau
Sent: Saturday, 30 January 2016 8:03 AM
To:
[hidden email]
Subject: Re: reflected light profilometry trrough plastic - mystery bands
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Looks like a lot of scatter off something in the sample. When you scan a nd look at the sample sitting on the stage do you see a lot of light off the sample? I mean by eyeball, not with the microscope optics. It's possible your glue is scattering and confusing your interfaces, also possible that the mylar is very scattering. You can also have lots of multiple reflections between surfaces, although the intensity of such 'ghost'
reflections tend to be much lower than the main reflection. Finally, what size confocal pinhole are you using, and have you tried other wavelengths?
Craig Brideau
On Fri, Jan 29, 2016 at 11:55 AM, Stanislav Vitha <
[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> Hallo,
> I have a little mystery.
> I am trying to measure depth of a microchannel (empty, air filled)
> using confocal microscopy in reflected mode.
> The microchannel is created by sandwiching a ~20 um PET (Mylar) spacer
> between two ~170 um thick Mylar sheets. The spacer has a thin layer of
> cyanoacrylate glue on both the top and bottom side.
> I am performing line-Z scan, with 0.25 um z-step, using a 20x/0.75 dry
> objective.
> I expected to get two reflections, one from the top of the channel,
> one from the bottom of the channel, just like it worked on
> microchannels made out of glass that I imaged previously.
> However, I am getting additional reflections and am not sure why and
> also do not know which one I should take as representing the bottom of
> the channel.
> See the image here:
http://i.imgur.com/Scj4i6y.jpg Reractive index of
> mylar is about 1.5, but I do not think I need to correct for z-axis
> compression since the channel is fille with air.
> The reflections are at 20, 40, and 60 um from the first reflection
> (top of the
> channel)
> The lineZ-scan through the spacer shows two reflection peaks, nominally
> ~40um apart, after z-axis correction it is ~60 um. Even if this is true
> and
> the spacer is really 60 um thick, I am still puzzled by the extra
> reflections in the air channel.
> Any hints/input is appreciated.
>
> With regards,
>
> Stan Vitha
> Microcopy and Imaging Cneter
> Texas A&M University
>