Posted by
Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/reflected-light-profilometry-trrough-plastic-mystery-bands-tp7584686p7584687.html
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Looks like a lot of scatter off something in the sample. When you scan and
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:
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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.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
>