Re: How to align polarization filters in two filter cubes

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-confocal-Z-stack-tp593216p593226.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I've been playing around with it a bit, and I've discovered that an LCD computer monitor makes a very good polarized "light box" that can be used to check the orientation of the filter once it's in the filter cube. I should pass on though that not all LCD screens emit polarized light in the same direction; one of our lab computer screens is horizontally polarized while my PowerBook Mac laptop screen is polarized at 45 degrees from the vertical.

John

On 5-Jun-08, at 3:36 PM, Barbara Foster wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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