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[Preconditions] [Acquisition Bleaching] [Imaging Steps] [Prebleach] [Bleach] [Postbleach]

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In the bleaching step one or more spot(s) or region(s) of interest (ROI) are irradiated with high intensity illumination.

Ideally the bleaching event should be instantaneous, in practice it should not exceed a tenth of the half time of the recovery. Therefore for analysing rapid kinetics, more powerful lasers as well as time optimized acquisition routines are essential.

Parameters that influence the bleaching process:

• Laser power: More laser power enables faster bleaching but also can harm the cells.
• Zoom: Zooming in increases the effective irradiation of the scanned area. Thus zooming in speeds up the bleaching, but the response time for switching back to the unzoomed imaging mode can delay the acquisition of the postbleach series. Which is especially undesirable when analysing rapid kinetics.
• Scan speed: The slower the scan speed the more energy is radiated (longer pixel dwell time)

It is important to calibrate the bleached volume for each set of parameters (laser power, objective, zoom, speed, etc.) which is best done using fixed samples. A more precise definition of the bleached volume along the optical axis can be achieved using two-photon excitation.

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contact: Stefan Terjung           last update: 02/06/04