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Standard features of typical CLSM necessary for FRAP experiments:
- AOTF to attenuate the laser power between high intensity illumination for bleaching and low intensity for imaging mode
- Interactive definition of the bleaching area by regions of interest (ROI) or spots including saving parameters of the ROI(s) or spot(s) for evaluation
- The software should be as flexible as possible to define prebleach-, bleach- and postbleach-settings either by special routines or by macro-programming, e.g.:
- number and frequency of frames
- scan speed and zoom factor
- laser intensity (AOTF settings)
Depending on the required resolution and the desired depth of bleaching, different objectives are suitable for FRAP experiments.
For homogenous bleaching in z-direction objectives with a lower magnification like a 20x 0.7 NA lens are suitable, but result in a lower resolution.
Higher resolution with increased light intensity in the focal plane but incomplete bleaching in z-direction can be achieved using lenses with a high numerical aperture like a 63x 1,4NA objective. The thinner the structure subjected to bleaching the higher the NA should be.
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