Recording GFPFusion Proteins in Living Spermatocytes by Confocal Microscopy

In this subsection, we describe a way to obtain a series of time-points each containing multiple sections observed by phase-contrast optics and fluorescence microscopy with a confocal microscope. This method is especially useful for following the behavior of cell structures such as the chromosomes or the spindle, by recording a-tubulin-GFP or Histone2-GFP (see refs. 18,21, and Fig. 5 of Chapter 2).

3.4.1. Recording a Multisection, Dual-Channel, Time-Lapse Series of a GFP-Fusion Protein

As explained in Subheading 3.3.1., the recording conditions for the microscope must be set up before starting the dissection.

1. Follow steps 1 and 2 of Subheading 3.3.1.

2. Switch on the lasers and initialize the software.

3. Select the objective from the software (see Note 17).

4. Set up the beam path by selecting the intensities and excitation wavelengths of the irradiated light and define the corresponding detectors used to record the image (see Note 18).

5. Choose the image resolution (we use 512 x 512) (see Note 19).

6. Specify the recording mode to be able to acquire a time lapse by scanning a stack of sections at each time point (we use the xyzt mode).

7. Follow steps 2-4 of Subheading 3.3.1.

8. Set the light source and change the microscope settings to the scanning mode.

9. Acquire in a continuous mode to be able to readjust the acquisition settings for the actual preparation (laser intensity, gain, offset, pinhole) in order to minimize irradiation and optimize the quality of the image (see Note 19).

10. Select the scanning speed (see Note 19).

11. Center the region of interest and zoom in to the desired degree.

12. Set origin and end of the series to be scanned at each time-point.

13. Set the number of series to be taken at each time-point and the number of accumulated frames per image (see Note 19).

14. Set up recording rate (number of time-points per minute). This will depend on the speed of the biological process to be followed (see Note 19).

15. Start acquisition. Record the cells during the time required, no more than 2 h or until signs of cell death start to appear (see Note 13).

16. Start a new series whenever refocusing or readjustment of the field is required.

17. Save the experiment (see Note 20).

3.4.2. Processing Multisection Time-Point Series

1. Two stacks (one per channel) can be built from the individual tiff images, using the NIH-Scion Image software as specified in Subheading 3.3.2. Each stack will display sequentially all the sections recorded at each time-point for the whole series of time-points (see Note 20).

2. Another possibility is to build stacks in which the information of the sections has been resumed. For the gfp channel, all of the sections can be projected at each time-point. For the phase-contrast channel, the optical section containing the focal plane of interest can be selected at each time-point (see Note 20).

3. In both cases two stacks (phase contrast and gfp) of the same dimensions and number of time-points will be obtained. Both stacks can be visualized and processed at the same time using Scion Image. Processing is similar to that described in Subheading 3.3.2. (see Note 16).

4. Merge the two processed phase and gfp stacks in order to visualize both at the same time.

5. Save the final merged stacks as movies in a format that is compatible with other computer platforms and image processing programs (see Subheading 3.3.2.)

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