Virtual spatially modulated illumination microscopy predicts nanometer precision of axial distance measurment

Abstract

Far field optical light microscopy with its unique capability for contactless, non destructive imaging inside thick transparent specimen such as cell nuclei has contributed widely to the present knowledge of the three- dimensional (3D-) architecture of the interphase nucleus. A serious drawback, however, is the limited optical resolution. A recently introduced light microscopical approach, Spectral Precision Distance Microscopy (SPDM) allows the measurement of distances between point-like fluorescent objects of different spectral signature far below the optical resolution criterion as defined by the Full Width at Half Maximum (FWHM) of the point spread function (PSF). Here, an aspect of the theoretical limits of this method was studied by virtual microscopy. The precision of the axial distance measurements was studied, taking into account photon statistics and image analysis. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, a precision of distance measurements in the nanometer range can be determined.