Method: Fluorescence Microscopy and Photography of In-Situ Slides

April 21, 1990

Rosalie Veile

This procedure is carried out on days 6 through 8 of the in- situ hybridizaton procedure.

Purpose:

To obtain photographs of fluorescent metaphases showing localization of cloned DNA sequences. DNA sequences homologous to the probe are detected with avidin-FITC (yellow color); metaphase spreads are distinguished with DAPI (4,6-diamidino-2-phenylindole dihydrochloride, blue color). The DAPI stain is used to identify chromosomes, which are photographed following the FITC photography.

Specialized equipment and supplies:

1. Zeiss Axiophot Photomicroscope equipped with epifluorence optics for DAPI and for FITC (located on 8th floor of McDonnell Science Building). Note: Must reserve time for use, by signing up on sheet located on door.
2. 63X Plan-Neofluar objective for UV excitation
3. 100X Plan-Apochromat objective
4. Zeiss emmursion oil and lens paper
5. 200 Watt Mercury Illuminator and power supply
6. 35 mm Camera with automatic exposure control
7. Ektachrome 64, 400 ASA film

Procedure:

1. Slides are stored in dark, plastic microslide boxes wrapped in parafilm at 4deg.C. Remove slides from 4deg.C and allow them to warm to room temperature.
2. Switch on the mercury lamp. The lamp must reach 200 Watts before using. Interrupt the beam path with the slider fully inserted (#1 of figure 1).
3. Using the room safelight only, remove a desired slide from microslide box and place on the microscope stage (#2 of figure 2).
4. Unblock the light path (middle position) with the slider (#1) and begin viewing the slide with the 20X objective. No oil is necessary with low power magnification. The reflector slider (#4) containing 3 filter sets should be used and positioned on the first position (blue filter or DAPI, see the drawing below).
   
5. Because a condenser aperture diaphragm will not affect the contrast, etc. in fluorescence (used in bright field and phase microscopy), only use the luminous field diaphragm. Refer to figure 1, lever (#2), and close the diaphragm to the point that an image becomes visible, center the image with (#3), and open the diaphragm until all chromosomes in metaphase are visible.
6. Using the coaxial coarse fine focusing control (#9, refer to figure 2), focus on the slide.
7. Begin scanning the slide for quality metaphase spreads using the coaxial control located below the right side of the stage. Metaphases should be as follows:

   1. Chromatids should be closely aligned.
   2. Chromosomes should not be overly contracted (>500 band stage preferred, i.e., high resolution chromosomes).
   3. There should be few crossed over chromosomes; chromosomes should be well spread but not so scattered that all are not visible within the same field of view.
   4. Chromosomes should be sharp and distinct. There should be no cellular debris or scratches on metaphase spreads (scratches remove DNA and interfere with the hybridization results).
   5. Background hybridization should be suppressed well with blocking solution (BSA).
   6. Red fluorescence created by Propidium Iodide stain (PI) and viewed with the FITC-green filter should be neither extremely bright , which could possibly mask the single copy signal (yellow), nor too weak, which may not be seen and/or would require extremely long exposure times in photographing (which would fade the fluorochromes).
   7. Single copy probe DNA (yellow doublet signals) should be seen in interphase nuclei.

      Special note: DAPI fluorescence is extremely bright and will quickly fade FITC fluorescence. Do not expose a metaphase to DAPI for more than 10 seconds prior to viewing it with the FITC filter. Care should be taken in protecting the image to be photographed by using the slider when work is interrupted and when switching to other objectives. Also, the intensity of illumination can be reduced by using neutral density filters.

8. When a desired metaphase is located, place one drop of Zeiss immersion oil onto the slide and switch to the 100X plan-apochromatic objective. This is done by turning the nosepiece counterclockwise.
9. Fine focus and center the metaphase within the shaded circle area using the lighted frame (reticle). By pressing the FRAME key on the camera control, the reticle will appear as a luminous frame. The brightness is variable to comply with the image brightness. This is accomplished by pressing down on the FRAME key until the desired brightness is obtained. The reticle is set when the FRAME key is released.
   
10. Using the 3 filter reflector slider, switch to the FITC filter (green).
    
11. Refer to figure 1, fine focus and prepare to photograph if the metaphase is desirable. Block the light path with slider (#1) when loading film.
12. Prepare for photography as follows:
    1. Remove the camera back/cassette from the microscope, and load 400 ASA film (figure 4).
    2. Set the ASA value to 400 (#3, see figure 4), which will automatically be transferred to the control panel.
    3. Mount the cassette by holding it on the sides. The film counter will automatically advance to O on the control panel (see figure 3, below).
    4. Compensation for reciprocity failure is achieved by pressing RECIPR key and the code number 6, then pressing the ENTER key on the control panel.

       e) Press 35L or 35R followed by ENTER to determine which camera port is used.

    5. Check the displays on the control panel for accuracy. If the values are not correct, review steps above.

       h) A green signal lamp reads OVER when the brightness needs to be corrected; and will read UNDER when the brightness needs to be increased.

       Figure 3: Camera Control Panel

       

    6. Exposure adjustment keys should be set on 0 and -1 at the start of photo taking (figure 3). Desirable exposure times are shown in Table 1. If the exposure time is > 200 seconds, decrease the time needed by moving to a lower exposure adjustment, such as -2 or -3. Then if the exposure time is still >200 seconds, the image can be photographed with the 63X neofluor objective. Note: Metaphases will differ in exposure times.

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    Table 1: Approximate exposure times of metaphases using FITC (green) filterfirst and DAPI (blue) second.

    Objective	Exposure Adjustment	FITC	DAPI
    63X	0	15-35 sec.	5-10 sec.
    63X	-1	0-20 sec.	0-05 sec.
    100X	-1	150-300 sec.	45-90 sec.
    100X	-2 or -3	0-150 sec.	30-45 sec.

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13. There are 2 beam splitters which need to be adjusted for observation and then for photography. See figure 5 and the diagram below.
    
14. After the beam splitters are adjusted for photography press the START key on the control panel and a picture will be taken. The film will automatically advance and the electrical counter on the control panel will continue counting (refer to figure 3).
15. Photograph first with the FITC filter, then switch to the DAPI filter. During photography, data such as the date, probe name, slide#, frame #, etc., should be written down (see table).
16. It may be necessary to scan slides a long time to find a metaphase of good quality. Adequate time should be alloted over several days to optimize the chances of photographing the most metaphases under the best conditions. Many variables affect the outcome of the quality of chromosomes:
    1. the chromosome preparation
    2. freezer location of slides (-80deg.C, not -20 deg. C)
    3. white blood cell count of donor of blood
    4. concentration of the blocking solution
    5. concentration of the stains (DAPI and PI)
    6. insufficient amount of human competitor DNA and/or salmon sperm DNA
    7. appropriate temperature of buffers
    8. appropriate time of washes
    9. quality of formamide (pH should be 7.0)
    10. slides must be scanned thoroughly

17. After the desired number of photographs are taken, rewind the film: Press the rewind button on the camera back (#5, see figure 5). Remove the camera back from the port and release the film.
18. Turn off the mercury lamp at the power supply. Record the number of hours the mercury lamp was used, the date, and person who used the microscope on the sheet of paper that should be next to the scope. (It is necessary to keep track of the total amount of time the bulb has been used; it must be replaced periodically because the quality of light degrades with use.)
19. Check all objectives for cleanliness. Remove oil with a quality lens paper and clean the stage with alcohol wipes or ethanol.
20. After the lamp power supply and illuminators have cooled (5-10 minutes), place the microscope cover over the scope to protect it from dust . Also replace the camera control panel cover.
21. After photography, in-situ slides can be stored at 4 deg. C for 2-3 weeks. The chromosomes may be G-banded and stained for chromosome identification (see chomosomes banding procedures).
22. Film can be developed into slides the same day at most photo labs. Marvin's Camera in Belleville or Art Tech photo lab in St. Louis is preferred.
23. After viewing the film slides of each probe hybridization, choose slides for 5" x 7" print enlargements of the FITC exposures and 3" x 5" prints of the DAPI exposures. Label the slides with the probe name, date, chromosome localization, magnification (optional), and initials of the preparer.
24. Analysis of film slides and the results should be recorded.

References:

Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and D. C. Ward. (1988) " Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries." Human Genetics 80: 224-234.

Cremer, T., Lichter, P., Borden, J., Ward, D. C., and L. Manuelidis. (1988) "Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes." Human Genetics 80: 235-246.

Lichter, P., Cremer, T., Tang, C. C., Watkins, P. C., Manuelidis, L., and D. C. Ward. (1988) "Rapid detection of human chromosome 21 aberrations by in situ hybridization." Proc. Natl. Acad. Sci. USA 85: 9664- 9668.

C. Zeiss (1989) Axiophot Photomicroscope Operating Instruction Manual.