In order to achieve a phase contrast image your microscope must be set-up with the proper phase contrast components. These components must be properly aligned and centered. Each objective must be centered to the corresponding phase annulus. In order to center for phase contrast please follow the instructions below.
1. If possible please align your microscope for kohler illumination.
2. Remove one eyepiece and insert the phase contrast centering telescope.
3. Focus the phase contrast telescope so that the phase plate and the phase annulus are sharp and in focus.
4. Start with the lowest magnification phase objective. If it is 10x Ph1 put this objective in place and the corresponding Ph1 phase annulus.
5. Observe the phase plate and the phase annulus through the phase telescope. While looking through you will see something like the image below:
Images a & c are an example of a system that is not centered and aligned for phase contrast. Image e shows a perfectly centered system. The segmented circle of light falls directly into the black ring.
6. In order to center your system if it is not aligned you will need to rotate the adjustment screws. Some condensers have these built into the condenser, typically two silver plungers sticking out of the condenser. Other systems require you to insert centering tools.
7. Once you have centered the first objective you can move onto your next objective.
8. Once complete remove the phase contrast centering telescope and re-insert the eyepiece.
Once your microscope is fully centered for phase contrast it should hold its position. There really should be no need to re-center unless the system is tampered with or it is a written protocol. You should check for proper centration periodically by inserting your phase centering telescope.