PM: Alignment Procedure

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polarized microscopes

In polarized light microscopy, proper alignment of the various optical and mechanical components is a critical step that must be conducted prior to undertaking quantitative analysis between crossed polarizers alone, or in combination with retardation plates and compensators. Several essential components must be correctly positioned with respect to both the microscope optical axis and to other mechanical and optical components. The series of alignment steps outlined below are intended for general use with polarized light microscopes and should be applicable to both student and research-level instruments. At a minimum, these microscopes must be equipped with two linear polarizing elements. One of the polarizers termed the Polarizer is placed beneath the stage inside or below the condenser, or covering the microscope light source exit port in the base. The second polarizer termed the Analyzer is installed above the objective rear aperture, either in a nosepiece slot or within an intermediate tube positioned between the frame and the observation tubes.

Many polarized light microscopes are equipped with a graduated circular stage that can rotate through 360 degrees for quickly changing specimen orientation with respect to the polarizer and analyzer transmission axes. In addition, one of the eyepieces in a polarized light microscope should have a crosshair reticule to identify the center and axes of the optical field. Advanced microscopes include a Bertrand lens for conoscopic observation of interference patterns focused in the objective rear aperture, but this accessory is not absolutely necessary for routine observations. Finally, polarized light microscopes should have an accessory slot in the nosepiece or intermediate tube into which a retardation plate or compensator can be inserted for quantitative analysis of birefringent specimens. Polarized Microscope Alignment Procedure The first step in polarized light microscope alignment is to establish proper Kohler illumination with both the polarizer and analyzer removed from the optical pathway.

Place a brightfield (stained) specimen on the stage and rotate the 10x objective into the observation position. Focus the specimen with the objective and close the field diaphragm iris to its smallest setting. Bring the partially closed field diaphragm into sharp focus in the same plane as the specimen by adjusting the condenser height. Use the condenser centering screws to translate the image of the field diaphragm iris opening to the center of the eyepiece crosshairs, and then open the diaphragm until the leaves are just outside the viewfield. Before inserting the polarizer and analyzer, the 360-degree circular rotating stage should be centered to the 10x objective so specimens will remain in the exact center of the viewfield when they are being rotated. Centering of the stage and objectives is also an important step for quantitative analysis of birefringence and optical path differences. A majority of the circular microscope stages pivot on three adjustable pins, one at the front of the stage for correct positioning and the other two equally spaced at 120-degree angles near the rear of the stage.

The stage can be centered with minute adjustments to the rear pivot pins by turning the centering screws. Start by locating a small circular feature on a relatively simple brightfield specimen in an area devoid of other distracting structures. Next, relocate the chosen feature to the center of the viewfield in the eyepiece crosshairs. Rotate the stage through 180 degrees until the centered specimen feature is overlapped by one of the vertical eyepiece crosshairs. Using the pivot screws, relocate the specimen feature to a distance approximately half way back to the crosshair center. Finally, move the specimen feature back to the center of the crosshairs and repeat the alignment sequence. After a few cycles, the specimen feature should be centered in the viewfield as the stage is rotated.

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polarized microscopes

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Saturday, May 19th, 2007 at 5:04 am

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Polarized Microscopes

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PM: Alignment Procedure

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