Lab Protocols:
Imaging protein mobility with PA-GFP
The mobility of HP1a measured using PA-GFP. (a) The mobility of PA-GFP-HP1a in the nucleus of a mouse GHFT1 cell was monitored following a 500-ms photo-activation pulse of 405-nm light at 135 uW laser power (circle), and the sequence of images show the diffusion of PA-GFP-HP1a throughout the nucleus; the calibration bar is 10 um. The diffusion rate was measured over a 50-s time frame in the indicated ROI (square). The diffusion rate was measured in 5 different cells, and the results are plotted in (b). Data first published in Demarco et al., 2006.
Confocal microscopy for photoactivation of PA-GFP - Timing 1-2 h

1. The cover glass with the monolayer of transfected cells is placed in the chamber, the insert is tightened snugly to the glass, and the chamber is filled with media. The chamber is then fitted to the prewarmed stage of the inverted microscope, and covered with the top from the 60 mm dish. The stage incubator maintains the temperature between 35-37ºC. For cultures that are exposed to room air, it is important to use a culture medium buffered to maintain pH under those conditions.

2. In order to optimize photoactivation of the PA-GFP labeling the proteins of interest, we co-transfect the cells with an mRFP marker to allow visualization of the transfected cells (see: Voss et al., 2004), which are identified using arc lamp illumination. The epi-fluorescence microscopy configuration on the Zeiss 510 confocal microscope is:

  • AttoArc 2 HBO, 100 W mercury lamp.
  • Neutral density filters to control illumination.
  • Standard FITC (ex 480/40 nm, beamsplitter 505 LP, em 535/50 nm) and Texas Red filters (ex 560/50x nm, beamsplitter 595 LP, em 645/75 nm) will work for this application.

3. Identify a transfected cell by mRFP fluorescence using the Texas Red filter, then switch to LSM mode. A power meter is used to directly measure the laser power at the specimen plane (Model SSIM-VIS & IR; Coherent, Inc.). Follow the link for the laser light path configuration; the initial laser power settings are:

  • 405: 1.5 uW (1% transmittance)
  • 488: 2-6 uW (1-5% transmittance)

4. The detector gain is initially set to 700, but this will be adjusted later as needed. The pinhole settings for each channel are initially set to 1 airy unit.

5. Using the red channel only, acquire a focused image of the selected cell.

6. Scanner configuration (speed, zoom factor): Using the Crop function, crop the image to the size of the cell. Set the maximum scanning speed possible for the selected zoom factor. The typical scanning speed for our experiments was 0.64 ms/pixel. Using the Histogram function as a guide, adjust the gain and the laser power so that the brightest pixel in the 8-bit image is between 200 and 250 gray levels.

7. Open a new image window, then turn on the green channel and do a preliminary scan of the cell.

8. Time Lapse Configuration: Under the Time Lapse function, set the number of scans and the delay between scans. This step must be optimized for the individual PA-GFP-labeled protein. For proteins that have very high intracellular mobility, set the delay to zero.

9. Defining the photoactivation spot: Under the Edit Bleach function, set the activation pulse length and intensity. As a starting point, set the number of iterations to 15, and the 405-nm laser to 100% transmittance. Set the number of images that are going to be taken before the activation pulse. Under Define ROI function, choose and draw the activation ROI. To avoid extensive photodamage, this ROI should be the smallest possible for efficient activation. We typically used a 20 pixel-diameter, circular ROI. Save the selected ROI for use in all future experiments.

10. Photoactivation: Place the photoactivation ROI in the area of the cell where PA-GFP is to be photoactivated. Return to the Time Lapse window and press Start Bleach.

11. Data analysis at the microscope: After activation and acquisition are done, the image sequence can be reviewed by choosing the Slice function. By choosing the Profile function, the change in fluorescence intensity over time in several different user-defined ROI can be determined. Then, by using the Show Table function, the actual intensity values for each selected ROI can be displayed and saved for later analysis.

12. Repeat these steps using different cells until photoactivation pulse is optimized.
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