After the stage has been centered with respect to the microscope optical axis with the 10x objective installed in the optical train, the other objectives (4x, 20x, 40x, and 100x) should be sequentially centered to the stage. Microscopes having centerable objectives contain a pair of Allen (or a similar drive) setscrews in the nosepiece that translate the objective laterally within its seat. Each objective can be centered using the procedure described above for the circular stage by using the objective centering tools provided by the manufacturer. Accurate centering of the stage and all objectives is paramount to quantitative analysis in polarized light, and avoids the operator losing sight of specific features in the viewfield during stage rotation. After completing the centering procedure, review the steps to ensure all of the objectives on the microscope are accurately centered before continuing. Establishing Kohler illumination and centering the microscope stage and objectives is easily accomplished in brightfield mode but can also be undertaken with a birefringent specimen in polarized light mode, if necessary.

To continue with alignment of the polarizing components, install the fixed or rotating polarizing element in the condenser or a housing on the light source exit port in the microscope base. Many polarized light microscopes are equipped with a fixed polarizer that has a transmission vibration axis oriented East-West, or left-to-right when standing in front of the microscope. Other microscopes have polarizers that can be rotated through 180 or 360 degrees, and are often mounted into frames with graduated increments ranging from 1 to 90 degrees. Ensure that the polarizer is correctly secured into position and either permanently fixed into its holder or placed in the zero position. The polarizer transmission vibration axis must be set to the East-West orientation in this step. Insert the analyzer into the microscope nosepiece or intermediate tube. Virtually all research-level polarized light microscopes have a rotating analyzer mounted in a rectangular frame that enables rotation of the element through 180 or 360 degrees.

Student microscopes often have a fixed analyzer as well as a fixed polarizer to ease the burden of crossed polarizer alignment. Rotate the analyzer while examining a birefringent specimen on the stage with the 10x objective. When the transmission axes of the analyzer and polarizer are crossed at a 90-degree angle, maximum specimen birefringence equivalent to maximum specimen brightness should be observed on a very dark background a condition that is termed maximum extinction. Under crossed polarized illumination, the vibration axis of the analyzer should be oriented North-South or front-to-back while standing in front of the microscope. A critical assessment for maximum extinction can only be accomplished while observing the background intensity through the microscope eyepieces. Keep in mind that at this point, maximum extinction only indicates that the polarizers are crossed, but does not guarantee that their transmission axes are perfectly oriented East-West and North-South. The latter aspect is addressed in the next step.