Posted by
Barbara Foster on
Jun 05, 2008; 7:36pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593225.html
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Hi, Jon
Your idea about putting the polarizer on top of the condenser is actually
a better one than to have both in filter cubes. I can't remember
the exact configuration of the filter slot there, but I would check to
see if it will give you enough room so that you could glue a small handle
to your polarizer and still rotate it, at least through 45 degrees (90
would be ideal).
As for the analyzer: a less elegant but perhaps more controllable
approach might be to remove the binocular body and install it in that
interface location, maybe with a small retaining ring to keep it in
place. Since the binoc fits on with a dovetail, that location might
give you the ability to align without disturbing when you "tighten
down".
Re: alignment. The ultimate test is full extinction when the two
polarizers are crossed. In an optics lab, you can test those positions by
shining light through the two into spectrometer. Obviously,
you need to calibrate one against a known first to learn its permitted
direction of vibration, then rotate the second until the intensity of
light emerging reaches a minimum.
One other caveat: you may want to test your objectives to see if
they de-polarize the light. Most of us in biology want high NA and
high resolution and therefore would probably prefer to work with
apochromats. Unfortunately, there are typically on the order of
17-21 lenselts inside, each of which is subject to strain which will
depolarize the illumination. There is also the issue of the glass
chemistry. Fluorites are often a better choice. I would
recommend that, when you are trying to find the fully crossed position
for your two polars, you do so with no optics in the way. That
will, essentially, provide a bare system response. Then you can
rotate each objective into the path and observe the intensity of the
background. If it goes from velvety black to charcoal gray, you know that
the optics are depolarizing the light.
By the way, there is a clever device that is about to come onto the
market called a "QLS" (Quantitive Light Source") that
might be helpful in the testing phase. Contact James Beach"
<
[hidden email]> (I've cc'd him on this response).
Good hunting!
Barbara
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At 12:57 PM 6/5/2008, you wrote:
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello
confocal listserver,
I have an unusual situation that I'm hoping someone out there might be
able to advise me on. I would like to modify my lab's inverted Olympus
IX70 microscope to have the ability to look at polarized light when
illuminated in the transmission pathway. I want to polarize the white
light that illuminates the sample, and then analyze the polarization of
the image by examining the light that comes through the sample with
another polarizer that can be oriented parallel and perpendicular to the
direction of the polarizer in front of the light source.
We have DIC optics for the microscope - this means that I have a
polarizer that can be fitted on top of the light source condenser, and I
also have a fixed position analyzer that can slide in below the
fluorescence filter cube turret. My problem is that the polarizer in the
analyzer slider is fixed in a direction that is crossed to the polarizer
in front of the light source. What I really want to do is to be able to
rotate the the polarizer in the slider to the parallel direction as well.
I know that Olympus and Leeds sells rotating analyzers that can slot into
these positions on the microscope, but my supervisor does not have the
funds to afford these right now. So I've come up with a slightly cheaper
alternative solution.
We bought two 1" round film polarizers from Edmund optics (~20$
each). These are the exact same size as interference filters that
normally get fitted into the fluorescence filter cubes which go in the
rotating turret below the objective nosepiece on the inverted microscope.
My plan was to take two empty fluorescence filter cubes and insert a
polarizer in each - one oriented in the parallel direction and one in the
perpendicular direction. Here is my problem: how can I orient and lock
the position of the the two polarizers in the filter cubes such that they
are aligned with their polarization axis directions correctly? Ideally,
since I'm trying to be quantitative with the imaging, I'd like their
alignment from their relative crossed position to be within 1 degree or
less. When I screw them down with the retaining ring of the filter cube,
they rotate slightly in the cube before they become locked. Also, I can't
tell if I'm dropping them into the filter holder in the right position
since their reference axis position is marked on the edge of the filters.
Can anyone out there suggest a good method to overcome this problem? How
do the microscope manufacturers align their polarizers in the sliders
they sell for DIC imaging, etc?
Thank you in advance for any help!
John Oreopoulos, BSc,
PhD Candidate
University of Toronto
Institute For Biomaterials and
Biomedical Engineering
Centre For Studies in Molecular
Imaging
Tel: W:416-946-5022