Setting up Nomarski

From Kimmel Lab Wiki

Setting up Nomarski optics on Zeiss Axiophot 2

Sign in with your name, date & time and whether you use the Mercury burner or not.

Select the objective and optivar appropriate to your prep. It might help to focus on your prep first at a lower magnification then move to your higher selected magnification. Set up Kohler at the beginning of the session, then set up phase contrast. Re focus and re-center the condenser and adjust the Nomarski for each new prep.

Kohler:

1. Set condenser ring to DIC I. Make sure polarizer under stage is set to “0”. Make sure condenser lens lever to the left under the stage hasn’t tilted the lens. Make sure all filters are out of the path of the illumination. (Neutral density filters at 100; filter wheel at white light setting, i.e. no label and Nomarski black slider prism out of the light path).
2. Focus on your specimen.
3. Close field diaphragm (black wheel closest to you, right side) all the way down. Focus hexagon of light to sharpness and stop right at the point of transition from red to green by raising or lowering the condenser using the knob on the side of the condenser. (You might have to move your focused specimen out of the way with the stage manipulators. Don’t change focus.) Center the hexagon using the two silver-colored centering screws located under the condenser. Open the field diaphragm again just until the hexagon disappears from the field of view.

Nomarski Phase Contrast:

1. Set the silver condenser polarizer ring to the proper setting for your objective: I for 10X, II for 20X, III for 40X.
2. Remove one ocular. Turn down microscope illuminator to about 2 “bars” from bottom so you don’t ruin your night vision. Look down ocular and close condenser polarizer ring (silver sub-stage ring) until circle of light is 60% of your field of view for Nomarski phase contrast (leave at 90% for Kohler).
3. Slide black bar prism in …OR… select “NOM” on the filter wheel.
4. Adjust Nomarski imaging by adjusting objective prism slider screw. (This should show you a dark image going to light and back to dark as you turn the screw if you have good Nomarski. Around the middle is a good place to start for most imaging.
5. Re-adjust the condenser prism ring as needed for fine tuning. Adjust the light intensity if needed.
6. Add neutral density (ND) filters as needed using the wheels on the lower right of the body of the scope.
7. Look at the monitor and adjust prisms and ND filters as necessary when taking a picture with the Axiocam. The objective prism adjustment can compensate for shininess in digitized image in monitor.

   Use the checklist on the inside cover of the logbook to make sure you leave the scope in the correct configuration for the next person. TURN EVERYTHING OFF Make sure to note time when you turn off the UV so the next person won’t turn it back on too soon. (EXPLOSIVE HAZARD). Be sure to wait until the illuminators are cool to the touch before covering equipment. Turn off monitors and Axiocam before covering equipment. Turn off air table.

   Cover the equipment after making sure NOTHING is still on and the power sources are all cooled down.

Using the mercury burner UV lamp for fluorescent imaging:

1. Turn on UV power source under the scope air table. (The rule for safety and for burner longevity: leave on for at least 2 hours, leave off 2 hours or until completely cool)
2. Note the time in logbook.
3. Use the light switch button with the icon of 2 light bulbs, a big one and a little one to switch between illumination sources: white, UV and both. Switch button is located next to illumination rheostat.
4. Select the proper filter for your signal using the buttons to the bottom back of the focus knob to change the filter wheel position.