Calibration of the polarizer and analyzer axis alignment can be accomplished by one of two methods. Many manufacturers provide a polarized light microscope orientation plate with their research-level microscopes to assist in alignment of the crossed polarizers. These plates contain a perfectly cut rectangular thin birefringent crystal having an optical axis that is parallel to the long edge of the crystal. The crystal is mounted flat on a microscope slide and designed specifically for use on circular polarized light microscope stages. When the long axis crystal is oriented at a 45-degree angle to the polarizer and analyzer transmission azimuths, maximum birefringence is observed in polarized light. Rotating the crystal towards the polarizer axis progressively reduces the level of birefringence until it is eliminated when the long axis is exactly parallel to the polarizer transmission azimuth and the horizontal eyepiece crosshair. At this point, the crystal birefringence is completely extinguished and should be indistinguishable from the background. Note that the outline of the crystal is artificial.

And is included only to demonstrate that the long axis is parallel to the polarizer azimuth. If the polarizer and analyzer are not positioned exactly in a East-West and North-South orientation then the long axis of the crystal will not be parallel to the eyepiece crosshairs when the birefringence is completely extinguished. In this case, rotate the long axis of the crystal until it is parallel to the horizontal microscope crosshair, and then rotate the polarizer until minimum birefringence is observed in the eyepieces. Next, rotate the analyzer until the crystal birefringence is completely extinguished. If a Bertrand lens or focusing telescope is available with the polarized light microscope, alignment of the polarizer and analyzer transmission azimuths can be performed using one of these optical components. Without a specimen on the stage, cross the polarizers and insert the eyepiece telescope into the observation tubes or swings the Bertrand lens into the optical pathway. Focus the telescope or Bertrand lens on the objective rear aperture.

A dark polarization cross can be observed at maximum extinction with brighter regions flanking the arms of the cross, which should be oriented vertically and horizontally. In most cases, the horizontal and vertical components of the polarization cross are wider in the center of the field and narrower at the periphery. Objective Rear Aperture in Cross Polarization Illumination The polarization cross geometry originates from the depolarizing effects of spherical lens surfaces in the condenser and objective, and may vary in size and shape depending upon the quality strain reduction or elimination of the lens systems. However, the presence of a polarization cross is normal in most microscopes and can not generally be considered an artifact. The cross should be centered in the viewfield when superimposed on the eyepiece crosshairsand appear perfectly aligned in a North-South and East-West direction. If this is not the case as illustrated in the polarizer and analyzer should be rotated in combination until the proper orientation is achieved.

As discussed above, perfect alignment of the polarization cross thus, the polarizers with a transmission axis at zero degrees is essential for performing quantitative measurements of birefringence azimuthal angles with or without a retardation plate or compensator in the optical pathway. The polarization cross reference calibration is also useful if a fixed polarizer or analyzer becomes misaligned in the mount. The final step in polarized light microscope alignment is to adjust the condenser aperture diaphragm so the bright outer regions of the polarization cross that are visible at the edge of the objective aperture are blocked. This step dramatically improves the extinction factor of the microscope optical system, and the field will appear maximally dark when viewed in orthoscopic mode without a Bertrand lens or focusing telescope. After the microscope has been aligned and the polarization axes calibrated, it is ready for quantitative analysis of birefringent specimens.