Application of 3D tracking, LED illumination and multi-wavelength techniques for quantitative cell analysis in digital holographic microscopy

摘要

Digital Holographic Microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Partially coherent light sources and multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the utilization of light emitting diodes (LEDs) as well as the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that LEDs and multi-wavelength techniques enhance the axial resolution in inspection of reflective surfaces and quantitative digital holographic cell imaging.