Posted by
Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584690.html
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Hi Stanislav,
I would expect two reflections outside the channel ([air - first mylar] and
[last mylar - air]) and four reflections across the channel (every air -
mylar inerface), unless your whole sandwich is glued to some glass support
or something...
This does not quite fit your measured profile, as the two reflection
(outside the channel) should be 360 um apart (without correction).
Furthermore, the [channel air - mylar] interface further from the objective
should not correspond (in depth) to any reflection outside the channel, the
air channel should appear as having different thickness than the mylar
spacer, due to different depth correction.
I can't see any of those in your images... Is your channel really air-
filled? Can you fill it with fluorescent solution and take a profile of the
fluorescence alongside with the reflection?
Best, zdenek
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
http://www.kcci.virginia.edu/tel: 434-982-4869
---------- Původní zpráva ----------
Od: Stanislav Vitha <
[hidden email]>
Komu:
[hidden email]
Datum: 29. 1. 2016 15:51:19
Předmět: reflected light profilometry trrough plastic - mystery bands
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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.jpgReractive 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"