The resolution optimization and sampling strategies in practical digital in-line holography (DIH) with spherical wave illumination are analytically studied by optimizing system parameters. Optimal parameters of holographic recording, including illumination wavelength, numerical aperture of the spherical illumination wave, source-sensor distance, object–sensor distance, and sampling parameters of the sensor, for achievable resolution are worked out. A formula is derived to guide the DIH system design in general cases. Different sampling strategies associated with corresponding reconstruction waves (plane wave and spherical waves with various curvatures) are analyzed. The reason why recording with spherical wave and reconstructing with plane wave works well in practice is explained in detail. We also present how to determine the reconstruction distance and magnification to reconcile the curvature difference. The analysis is carried out mathematically and verified by simulated holograms. Optical experiments with U.S. Air Force resolution target are carried out based on the analysis for further verification.

The label-free analysis of leukocytes by holographic microscopy has the potential to overcome the extensive labeling procedures of conventional analysis by automated hematology analyzers and manual blood smear analysis. For the enrichment of leukocytes, selective lysis of erythrocytes is applied in flow cytometry to minimize the background. In the case of quantitative phase measurements, lysis procedures may affect the morphology of leukocytes inducing refractive index changes, which can deteriorate the specificity and sensitivity of the method. To study the effects of selective erythrocyte lysis on phase measurements of leukocytes, we compared two widespread methods: lysis by hypotonic shock followed by magnetic depletion of erythrocytes and lysis with a commercial lysis buffer containing ammonium chloride and potassium bicarbonate. We observed that lymphocytes were less affected than granulocytes and monocytes by both methods and that the intracellular changes increased with increasing incubation times. These findings indicate a significant effect of erythrocyte lysis on leukocytes and need to be studied for future hematology applications.