Image acquisition and calibration methods in quantitative confocal laser scanning microscopy

Quantitative measurements in far field light microscopy are complicated by the different lateral and axial resolutions. For principle reasons the spatial resolution in the direction of the optical axis is lower than in the focal plane. To overcome these limitations, we have developed a 2(pi) -tilting device for full specimen rotation perpendicular to the optical axis. Due to the influence of specimen and mounting media on the spatial resolution of a CLSM, the focal shift increases with the refractive index mismatch and the depth of the investigated region. Attenuation and absorption effects of excitation and emitted light due to layer thickness and refractive index mismatches have to be considered. By means of a capillary with a square shaped cavity in combination with the tilting device, it may become possible to directly calibrate the confocal system in the direction of the optical axis. With this technique it is possible to test 3D deconvolution and segmentation procedures applied to the same object acquired under different perspectives.

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