Self-calibrating lensless inline-holographic microscopy by a sample holder with reference structures

A self-calibration technique for lensless compact chip-microscopes based on inline holography with pinhole illumination is presented. The pinhole illumination wave acts as reference and is needed for the reconstruction process. This reference wave is assumed to be spherical, so that its phase is already determined by the position of the pinhole in relation to the image sensor. It is shown that the reconstructed spatial resolution is strongly dependent on the estimation for the pinhole to sensor distance. A precision in the range of tens of microns was reached for microscopic imaging with a spatial resolution in the range of one micron. Therefore additional reference crosses are prepared lithographically on the sample holder. The hologram, which contains the optical information about the sample as well as the reference crosses, is used for calibration and image reconstruction at the same time. The presented technique was tested to allow the reconstruction of a spatial resolution corresponding to the limit of detection apertures of about 0.66. The technique was applied to holograms of test beads and blood smear samples.