Single-molecule microscopy allows for the investigation of the dynamics of
individual molecules and the visualization of subcellular structures at high spatial resolution.
For single-molecule imaging experiments, and particularly those that entail the acquisition of
multicolor data, calibration of the microscope and its optical components therefore needs to
be carried out at a high level of accuracy. We propose here a method for calibrating a
microscope at the nanometer scale, in the sense of determining optical aberrations as revealed
by point source localization errors on the order of nanometers. The method is based on the
imaging of a standard sample to detect and evaluate the amount of geometric aberration
introduced in the optical light path. To provide support for multicolor imaging, it also
includes procedures for evaluating the geometric aberration caused by a dichroic filter and the
axial chromatic aberration introduced by an objective lens.