Identification of acquisition parameters from the point spread function ofa fluorescence microscope

Except for blind methods, deconvolution of 3-D data sets acquired from a fl uorescence microscope requires the knowledge of the point spread function ( PSF) of the instrument. Using the XCOSM package, we show first with simulat ions and then with recorded data that it is possible to recover from an exp erimental PSF some parameters, which are very difficult or impossible to me asure during the acquisition, like the specimen depth or the immersion medi um refractive index. Doing so, we can precise the acquisition protocol, whi ch helps to use the instrument under optimal conditions. Furthermore, the k nowledge of the actual acquisition conditions permits to use for the deconv olution process a computed PSF, which is noiseless and as close as possible to the actual PSF. This helps to reduce errors in quantitative measurement s after deconvolution, as shown with computations. (C) 2001 Elsevier Scienc e B.V. All rights reserved.