There are several physical-mechanical requirements. The sub-stage condenser must be capable of being focused up and down, preferably by a knob operating on a rack and pinion. The sub-stage condenser must be fitted with an aperture iris diaphragm that can be opened and closed by a lever or knob. The lamp must be fitted with a condensing lens, a collector, and a field iris diaphragm that can be opened and closed. It is also desirable that the lamp filaments or bulb be centerable or pre-centered. To repeat, there are two important adjustable iris diaphragms; the aperture diaphragm at the sub-stage condenser and the field diaphragm nearer to the lamp. The aperture iris diaphragm controls the angular aperture of the cone of light from the condenser. The field iris diaphragm controls the area of the circle of light illumination the specimen.

Step 1. Open the aperture iris diaphragm wide and also open the field iris diaphragm wide. Turn on the lamp. Using a low power objective (10x or so) and a 10x eyepiece, slowly focus the specimen that has been placed on the microscope stage over the stage opening.

Step 2. Close the field iris diaphragm most of the way. Using the rack and pinion knob of the condenser, raise the condenser until the edges of the partly closed field iris diaphragm appear superimposed on the already-focused specimen. The edges of the field iris diaphragm should appear sharply focused. The substage condenser is usually close to its highest position.

Step 3. If the field diaphragm does not appear centered in the field of view, use the substage condenser centering screws to center the field diaphragm. Then slowly open the field iris diaphragm until it just disappears from view. This step must be repeated each time a different objective is turned into place on the nosepiece.

Step 4. Now lift the eyepiece out of the body tube and look down the tube at the back lens of the fully-lighted objective. (This is best accomplished by the use of a pinhole eyepiece – an eyepiece with a tiny hole but no lenses or a focusing telescope such as is provided for use in phase – contrast microscopy.) while looking down the microscope tube, slowly open and close the substage condenser aperture iris diaphragm. It will be seen that closing the aperture iris diaphragm "cuts into" the periphery of the back lens of the objective. For excellent illumination and contrast, approximately ¼-1/3 of the back lens should be occluded, thus leaving 2/3-3/4 of the back lens illuminated. Then replace the eyepiece in the tube. This step too must be repeated each time a different objective is turned into place on the nosepiece.

The aperture iris diaphragm may have a calibrated scale (sometimes the calibration refers directly to numerical aperture) which can be used to make the iris adjustment more accurate as well as readily repeatable.