High-resolution mapping of the three-dimensional point spread function in the near-focus region of a confocal microscope

Fluorescent nanobeads with a diameter of 20nm were used to map the three-dimensional point spread function in the near-focus region of a confocal microscope at high spatial resolution. Fluorescence images were taken in 109 equidistant planes (50nm apart) parallel to the focal plane; postacquisition stacking of these images allows the reconstruction of the point spread function in the axial plane. The experimental distribution is compared to theoretical calculations based on an integral representation for the light intensity in the focus region that takes into account stratified media, polarization, the Gaussian illumination profile, and the finite exit pinhole size.