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

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The prebleach series is used to:

• determine the (total) fluorescence intensity at low intensity illumination prior to bleaching to provide a reference point for fluorescence recovery; typically 3-10 images.
  (If fluorescent proteins (FP) are imaged with more than 1 image/s a prebleach series of 50-100 images is needed to reach a steady state of FPs in dark states (Weber et al. 1999)).
• calculate the fluorescence loss due to acquisition photobleaching

To prevent artefacts from pixel saturation, the maximum intensity (255 in 8-bit images) should be reached only in very few pixels. The offset should be adjusted that the background pixels show grayvalues slightly above zero (otherwise information can be lost). To increase the dynamic range it can be advantageous to employ the 12-bit mode.

In the pre- and postbleach-series laser intensity should be attenuated as low as possible to get a sufficient signal - for FRAP experiments minimized acquisition photobleaching is more important than ‘nice images’.

In the data evaluation step the fluorescence intensity during recovery will be normalized with the prebleach values.

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