Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 101

coherence length . 1 m) or by a diode laser ( λ 5 641 nm, coherence length 0.75 mm). The

angle between the two mirrors M1 and M2 was chosen in such a way that a spatial carrier

fringe frequency with a spatial phase gradient near 0.5

α

per pixel [58] for optimized
numerical reconstruction was generated. The captured holograms are shown in Figure 6.4D
(frequency-doubled Nd:YAG) and Figure 6.4G (diode laser). The resulting parallel carrier
fringe pattern is depicted in the enlarged part of Figure 6.4D . Figure 6.4E and H shows the
spatial frequency spectra of the holograms in Figure 6.4D and G that were calculated by a
two-dimensional (2D) fast Fourier transformation. The narrow peaks that are marked in
Figure 6.4E and H with arrows show the high linear degree of the carrier fringe patterns.
Finally, Figure 6.4F and I presents the quantitative phase contrast images obtained by spatial
phase shifting reconstruction as described in Section 6.3.2 . Each of the two sheared wave
fronts serves as a reference wave for the complementary wave. Thus, the cells appear in
inverse phase contrast as they are imaged with different mirrors. For both light sources, the
frequency-doubled Nd:YAG laser and the diode laser, phase contrast images with a similar
quality were obtained.

π

A further characterization of the Michelson interferometer approach and comparative
investigations to a modular DHM system based on a Mach Zehnder interferometer with a
fiber optic reference wave (see detailed description in Ref. [27] ) were performed using the
frequency-doubled Nd:YAG laser as light source. Due to the small OPL difference within
the interferometer for the setup in Figure 6.2 , an up to five times increased temporal phase
stability comparison to the fiber optic Mach Zehnder setup was achieved [35] . The
increased interferometric stability also results in a significant reduction of reconstruction
artifacts like unwrapping errors which is important for the automated evaluation of digital
holograms obtained from time-lapse series.

However, the analysis of the spatial phase noise within the quantitative phase contrast
images also shows that the accuracy for the detection of OPL changes for the Michelson
interferometer-based DHM arrangement is lower than for a Mach Zehnder setup [35] . This
can be explained by the circumstance that in the Michelson interferometer setup both waves
are affected by coherent disturbances due to scattering effects from the microscope imaging
system and the medium in which the specimen is embedded. Nevertheless, in Refs. [35,36] ,
it is demonstrated that even subcellular structures like the nuclear envelope and the nucleoli
are resolved. Furthermore, the setup is particularly suitable for low-cost light sources with
short coherence lengths l c , 1mm [36] .

6.3.4 Subsequent Refocusing and Autofocusing

In digital holography, refocusing can be performed numerically by variation of the
propagation distance Δz in Eq. (6.2) . The combination of this feature with image sharpness

quantification algorithms yields subsequent autofocusing (for an overview, see Ref. [54]