Method and devices for measuring distances between object structures

Abstract

The invention concerns a method and devices for far field microscopy and flow fluorometry for geometric distance measurements between object structures, i.e. measurement structures, marked with fluorochromes, whereby the distances can be smaller than the half-intensity width of the principle maximum of the point spread function. In this method, the measurement structures are marked with fluorescent dyes with different or identical spectral signatures, according to their distances. Calibration targets with defined dimensions and arrays are marked with the same fluorescent dyes. Calibration targets and measurement structures are prepared separately or together on an object support and investigated microscopically or flow fluorometrically. In each case, two defined calibration targets with different spectral signatures are measured in consideration of the wavelength dependent imaging and localisation behaviour of the optical system used, the measurement values thus obtained are compared against the previously known real distance values and the difference is used as the calibration value for correcting the shift in the direction of the measurement structures caused by the optical system. In the case of the devices, these are calibration targets and an axial tomograph for undertaking the method described above.