We present a new detection method for multifocal two-photon laser scanning microscopy (TPLSM) that allows a fast and easy access to spectrally resolved, three-dimensional images. In our setup eight fluorescent foci are directed through a descanned tube lens combination and a straight vision prism. This prism spectrally splits up the fluorescence beamlets, resulting in eight parallel spectral fluorescence lines. These lines are imaged onto a slit block array in front of a 8×8 multi anode PMT. Each PMT row detects different spectral characteristics from a special point in the sample whereas each column represents one focus. The eight exciting foci are scanned in the region of interest inside the sample by the two scanning mirrors in x- and y-direction. As a result of this imaging technique eight spectrally resolved images of slightly shifted sample regions are generated simultaneously and added up after the measurement, maintaining the spectral information. We present spectrally resolved 3D-data of various biological samples like pollen grains, tobacco cells and orange peel cells.
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