Confocal laser-scanning fluorescence microscopy: In situ determination of the confocal point-spread function and the chromatic shifts in intact cell nuclei

The confocal point-spread; function (PSF) was measured in three-dimensional ly (3D-) conserved nuclei of human cells using point-like fluorescence labe ls coupled specifically to small chromatin regions. Since the resolution of a confocal fluorescence microscope, that is the smallest routine ly measur able distance using one spectral signature (fluorochrome) only, is normally given by, one full width at half maximum (FWHM) of the PSF, the results in dicate that distance measurements in cell nuclei under: tbe suboptimal opti cal conditions relevant here; are usually :limited to a regime Of about. gr eater than or equal to 0.30 mu m in lateral and greater than or equal to 0. 70 mu m in axial directions. Using two or more spectral signatures. however ,:ito-label point-like objects, the technique of Spectral Precision Distanc e Microscopy (SPDM) allows the :determination of distances below the above mentioned resolution criterion. SPDM requires the exact determination of ch romatic shifts under :optical conditions close to the actual condition used in the precision distance measurements. Here, high precision chromatic shi ft measurements insitu in cell nuclei are presented, opening the avenue to distance measurements far below the normal resolution limit. Thus,, for obj ects located in the interior of intact cell-nuclei, a "resolution equivalen t" of some tens of nanometers can be realised.