Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 99

Figure 6.3A shows a digital hologram of human red blood cells (RBCs) in phosphate
buffered saline. The cells were recorded slightly defocused with a 63 3 microscope lens
(numerical aperture (NA) 5 0.75) with a setup as shown in Figure 6.1 by using a frequency-
doubled Nd:YAG laser ( λ 5 532 nm) as light source. The enlarged area in Figure 6.3A

shows a part of the carrier fringe pattern of the digital off-axis hologram that was used for
holographic coding of the object wave. Figure 6.3B shows the unfocused reconstructed
amplitude image of the RBCs in the CCD sensor plane. Figure 6.3C displays the sharply
focused amplitude after numerical refocusing with Eq. (6.2) . This image corresponds to a
microscopic bright-field image under coherent illumination. The quantitative phase image
after removal of the 2 ambiguity is shown in Figure 6.3D , while Figure 6.3E shows a

corresponding pseudo-3D representation of the data. In Figure 6.3F , the cell thickness
measurement along a cross-section through the phase data along the dashed line in
Figure 6.3D is illustrated. The thickness values d cell of the RBC were calculated from the
phase contrast

π

Δϕ

cell with Eq. (6.4) by estimating an integral cellular refractive index
n RBC 5 1.400 [55] . The refractive index n medium 5 1.337 of the buffer solution was obtained
by an Abbe refractometer. For completeness, Figure 6.3G shows a DHM differential phase
contrast image (DHM DIC image) that has been obtained by the calculation of the

5 μ m 5 μ m

6.4 rad

0

(A) (B) (C) (D)

5

2.5 5 μ m

4
3
2
1
0

2.0
1.5
1.0
0.5
0.0

(E) (F) (G)

0246
x (µm)

8 0

Figure 6.3
Evaluation of digital off-axis holograms by spatial phase shifting. (A) Slightly defocused recorded
digital hologram of human red blood cells, (B) reconstructed amplitude in the hologram plane
(defocused), (C) numerically refocused amplitude distribution, (D) unwrapped phase distribution
coded to 256 gray levels, (D) pseudo-3D representation of the phase distribution in (E), (F)
cross-section through a cell along the dashed line in (D), (G) first derivative of (E) in the
x-direction. Source: Modified from Ref. [54] .