Tissue Culture Laboratory (BIOE342) - Protocol
Index



Zeiss Fluorescent Microscope

 

Set-Up

1.      Microscope and power supply plugged in and turned on (done by Instructor or T.A.).

2.      Top filter pushed to left.  Viewing through no filter.

3.      Mid-level filter in center.  Viewing without phase contrast.

4.      Aperture lever far to right.  Aperture is open.

5.      Light intensity set between 2 and 3.

6.      Magnification 10x or 20x.

7.      Fluorescent filter cube options:

·        GFP Filter: see FITC, Calcein, and Ethidium.

Excites at 470 nm;  emits at 500 nm long pass.

Filter cubes pushed to left. View through GFP (yellow) cube on right-hand side.

·        FITC Filter: see FITC, Calcein.

Excites at 470 nm;  emits at 535 nm.

Filter cubes pushed to right. View through FITC (green) cube on left-hand side.

·        Filter cubes in centerline.  View through neither cube, which is clear brightfield.

8.      Fluorescent light pass options:

·        Black lever positioned to block fluorescent light for brightfield viewing.

·        Black lever positioned to let fluorescent light pass for fluorescent applications.

 

Operation

1.      Turn on power on front of power source box (done by Instructor or T.A.).

2.      Place cells on stage.

3.      Turn off overhead lights, if needed.

DO NOT FIDDLE AROUND WITH BUTTONS NOT DESCRIBED TO GET THE MICROSCOPE TO “WORK.”  PLEASE ASK FOR HELP.

4.      Focus in on cells of interest using brightfield imaging:

·        Close fluorescent light pass.

·        Push filter cubes to center to see through neither filter cube.

·        Turn on power/light to microscope.

·        Focus on cells using gross and fine focus controls.

5.      To see through FITC filter:

·        Turn off power to microscope.

·        Push filter cubes to right to see through left-hand side cube.

·        Open fluorescent light pass.

6.      To see through GFP filter:

·        Turn off power to microscope.

·        Push filter cubes to left to see through right-hand side cube.

·        Open fluorescent light pass.

7.      To see through brightfield:

·        Close fluorescent light pass.

·        Push filter cubes to center to see through no filter cube.

·        Turn on power/light to microscope.