Figure 1 shows a FRAP experiment performed with a 63x lens at 1,400 Hz and a circular ROI with a diameter of about 5 μm. The zoom is increased from zoom 6 to zoom 30 to match the field of view to the 5 μm ROI for bleaching. A zoom factor of 5 delivers about 25 more power per area. With the conventional scanner, the full effect of Zoom In can be used. In the case of the resonant scanner, one has to take into consideration that the resonant x scanner cannot zoom in and out very fast. This is the reason why the Zoom In for the x scanner is blocked in the FRAP wizard. In consequence, even the maximum available laser power is often insufficient to bleach effectively. To compensate for the x scanner’s inability to zoom fast enough we introduced the FRAP Zoomer for the SP8. The FRAP Zoomer uses the y scanner for zooming in, while the x scanner retains its scanning angle. Thus, Zoom In can be applied for bleaching within FRAP wizard and RS at least for one dimension.
To compensate for the low bleach efficiency caused by very fast scanning it is helpful to change the fill factor of the back aperture of the objective. This can be done by retracting the beam expander optics (FRAP Booster) within the FRAP wizard. In the first step the beam expander can be retracted for the whole FRAP sequence (Figure 2).
If this option is active, the beam expander is retracted from the beam path. As a result, the back aperture of the objective is not completely filled with light anymore: The amount of light is the same but concentrated to a spot in the center, and about 2 to 5 times more light is available, depending on the objective (Figures 3–5).
Fig. 5: Left: without FRAP Booster. Right: with FRAP Booster. The same laser settings were used for both images. GFP-Tubulin in HeLa cells, EMBL Heidelberg.
Figures 6 to 8 are showing the results of FRAP experiments with the resonant scanner:
- Figure 6: Without FRAP booster, without FRAP Zoomer
- Figure 7: Without FRAP booster, with FRAP Zoomer
- Figure 8: With FRAP Booster and with FRAP Zoomer
HeLa cells with free YFP were used for the experiments. The FRAP series were taken with a 40x 1.1 lens and zoom factor of 8 was used during the whole experiment. The frame rate was about 36 fps, the bleaching time about 200 msec. If you need to bleach even faster, you may use all Ar-laser lines for bleaching.
It can be summarized that during FRAP experiments with RS it may happen that the time to apply the needed laser power is too short to bleach the region of interest efficiently. Then appropriate adjustments are needed to concentrate the available amount of light to a smaller area: The best bleaching results can be achieved by combining the FRAP Zoomer and the FRAP Booster.