Holographic Motility Contrast Imaging of Live Tissues - Biomedical Optical Phase Microscopy and Nanoscopy - page 206

This chapter describes the technology and underlying biological physics of holographic
motility contrast imaging (MCI) of live tissue. MCI is a statistical optics form of phase-contrast
imaging [6] . It grew out of holographic optical coherence imaging (OCI) that uses a
holographic coherence gate [1,7] to capture full-frame sections inside tissue of up to 1 mm
thick. Multimode illumination of the tissue samples generates highly developed speckle that is
sectioned in three dimensions based on optical path length. In live samples, the speckle is
highly dynamic, and subcellular motions provide an imaging contrast that differentiates the
outer proliferating shell of a tumor from its hypoxic or necrotic core. Changes in the motility
contrast and in the fluctuation frequency content caused by applied xenobiotics may provide a
new form of high-content drug screening for early drug discovery.

11.2 Optical Coherence Imaging

OCI is a full-frame form of optical coherence tomography (OCT) [8] . It uses holography both as
the coherence gate for detection and as a means to perform spatial demodulation and image
reconstruction. OCI is characterized by high-contrast speckle because of the wide-field
illumination of many spatial modes that self-interfere. The speckle size is also the spatial
coherence length, and a temporally coherent reference wave interferes with the speckle field to
produce interference (holographic) fringes within each speckle. These fringes enable coherence-
gated detection of path-matched light originating primarily from a specified depth in the tissue.
Therefore, understanding the performance of OCI requires an understanding of the speckle field
properties, the role of scattering, and the coherence gate in the presence of multiple scattering.

The optical setup for the OCI system is shown in Figure 11.1 . The probe beam is from a
Superlum superluminescent diode system with a wavelength of 840 nm and a bandwidth of
50 nm. The beam size at the target is about 1 mm. The target is a multicellular tumor
spheroid that can grow as large as 1 mm in diameter, but we typically work with tumors of
diameters around 0.5 mm. The backscattered light is collected through a polarizing
beamsplitter and a first Fourier lens L1. A second lens relays the image to the first image
plane IP1 which is the object plane of the Fourier transform lens L3. The signal arm passes
through a beamsplitter where it is joined by an off-axis reference beam with a crossing
angle of about 1.3 . The reference and signal beams combine on the charge-coupled device
(CCD) chip with a fringe spacing of approximately 25 μ m.

The speckle diameter at the CCD camera depends on the Fourier optics of the lenses and
apertures, shown in Figure 11.1B . For a target diameter D Samp , the speckle diameter at the
first Fourier plane FP1 is given by:

4
π

f 1 λ
D samp

d FP1
c

5

(11.1)