Confocal-FRAP Analysis of ECM Molecular Interactions - Methods in Molecular Biology: Extracellular Matrix Protocols - page 88

7. Owing to the high inertia of the scan head mirror assemblies found in some con-

focal microscopes, the scan-beam dwell time can be longer at the edge of a scan
than the center, leading to uneven bleaches, especially of rectangles. Before pur-
chasing a confocal microscope for confocal-FRAP, the user should ensure that
this problem does not occur. The MRC-1000 instrument is not prone to this effect.

8. To allow the collection of extended image series, a minimum computer RAM

size of 32 Mb is recommended.

9. At short displacements from the bleach center (<10 pixels) the total signal is low

and also pixelation means radial positions cannot be accurately defined. How-
ever, the variance calculation is insensitive to bleached fluorophore concentra-
tion near the bleach center, and these effects do not constitute significant errors.
Conversely, variance calculations are very sensitive to bleached fluorophore con-
centrations distant from the bleach center. The accurate measurement of fluores-
cence intensities at such positions is thus very important.

Acknowledgments
We are grateful to The Wellcome Trust for support.

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