Recording Living Spermatocytes by Phase Contrast Microscopy

In this subsection, we provide the necessary tips to acquire and process a time-lapse series of meiosis by phase-contrast microscopy using some basic image acquisition equipment. By following these steps, nice videos of Drosophila male meiosis can be obtained, as shown in refs. 18,20, and 21, and Fig. 5 of Chapter 2.

3.3.1. Recording Single Section Videos by Phase Contrast Microscopy

No time must be wasted once the spermatocytes are prepared within the culture chamber, because their life-span in primary cultures is short. Therefore, before dissection, it is important to already have done steps 1-4.

1. Adjust the recording temperature to 25°C at least 1 h in advance.

2. Make sure that the optics for phase-contrast microscopy is properly adjusted.

3. Check the shutter and shutter-driver connections.

4. Start the Scion-Image software and set up the acquisition mode.

5. Place the testes preparation under the microscope and find the region of interest (see Note 7).

6. Select the cells that are going to be followed (see Note 8).

7. Readjust phase contrast (see Note 9).

8. Adjust the illumination conditions (see Note 10).

9. Specify the recording rate depending on the process to be followed (see Note 11).

10. Restart the timer to 0 (see Note 12).

11. Record the cells during the time required, no longer than 2 h or until the first signs of cell damage start to appear (see Note 13).

12. While recording, pay attention to the focusing and repositioning of the cells within the field (see Note 14).

13. Save the images (see Note 15).

3.3.2. Processing Single Section Time-Lapse Series by Phase-Contrast Microscopy

1. Open all of the single images within the NIH-image software and make a stack. This can be animated and visualized as a movie at the desired speed following the instructions indicated in the software.

2. Process the stack so that the cells and the processes of interest can be followed. This includes brightness and contrast adjustment, alignment of the cells, and cropping of the desired region (see Note 16).

3. Save the modified stack as a movie in a format that is compatible with other computer platforms and image processing programs. QuickTime and AVI are the most commonly used.

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