Three-dimensional (3D) quantitative imaging of structures and dynamics of biological specimen such as cells could find numerous applications in cell biology and medicine, since it enables quantitative study on the specimen in various environments. Many 3D imaging techniques to date, however, require biological specimens to be stained or dyed to produce images of structures that are normally not observable, which may perturb biological functions.
Conventional phase imaging techniques such as phase-contrast (Zernike) or differential interference contrast microscopes produce high-contrast images of transparent specimens in their natural state without previously being killed, fixed, and stained. Thus, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail. Yet, the images obtained with those techniques cannot be directly used for quantitative studies, and are limited two dimensions, since they provide an image of a transparent object in transmission.
Our laboratory developed a novel 3D imaging modality referred to as optical coherence phase microscopy (OCPM) for non-invasive quantitative 3D phase contrast imaging. OCPM is based on the features of spectral-domain optical coherence tomography (SD-OCT), and produces depth-resolved intensity and phase images with significantly improved phase stability compared to the time-domain OCT based systems. Since SD-OCT acquires depth-resolved information without mechanical scanning of the reference mirror, it can generate a three-dimensional, quantitative phase contrast image of a specimen, simply by scanning the beam laterally as it measures phase information in depth.
Phase stability in OCPM determines the ability of the system to detect minute phase changes spatially and temporally. In order to quantify the OCPM phase sensitivity, we measured the phase fluctuation in the interference between top and bottom surfaces of a coverslip, and achieved phase stability corresponding to a resolution of 25 picometer, which demonstrates remarkable capability in studying biological processes.
An initial demonstration of the OCPM imaging performance was done by imaging an “MGH” patterned coverslip. For imaging, the phase in the interference between the reflections from the top and bottom surfaces of the etched coverslip was measured, as the beam scans laterally. Shown in Figure 1 are the images of the sample recorded by Nomarski microscope (10 ´, NA=0.3) and OCPM (NA: 0.2). Besides the improved contrast in the OCPM image, the gray scale to the top of OCPM image represents the depth with reference to the flat (not etched) surface in nanometers, which agreed well with the surface profiler measurement within 5 nm. Figure 1(c) shows the etch depth profile of the coverslip in 3D representation.
Lymphoblast cells were also imaged to assess 3D imaging capability of OCPM. The cells were placed between a microscope coverslip and a glass slide, and we obtained the depth-resolved information by measuring the interference signal referenced to the top surface of the coverslip. Figure 2 shows the images of two depth planes in the cells. The detailed description of the images can be found in the figure captions. Cellular and subcellular structures can be observed in the images, and the optically thick nuclei in the cells are clearly visible in the phase-contrast images.
Figure 1. Images of “MGH” patterned coverslip. (a) Image recorded by Nomarski microscope (10 ´ , NA: 0.3). The solid bar corresponds to 125 m m. (b) Image taken by OCPM. Gray scale represents the etch depth in nanometers. (c) Three-dimensional etch depth profile of the “MGH” patterned coverslip.
Figure 2.Images of lymphoblast cells. The intensity, phase-contrast, and phase difference variance images are shown for a plane at ~8 μm below the top membrane of the cells (a, b, c), respectively, and for a plane at the bottom of the cells (d, e, f), respectively. The intensity images (a, d) show the cellular and subcellular structures in the cells, while the optically thick nuclei are clearly visible in the phase-contrast images (b, d). The color scale bar to the right in the phase-contrast images denotes the optical path-length in air. The phase difference variance images (c, f) show the magnitude of phase changes in a two-dimensional plane.
C. Joo, T.A., B. Cense, B. H. Park, and J. F. de Boer, Spectral-domain optical coherence phase microscopy for quantitative phase-contrast imaging Optics Letters, 2005. 30: p. 2131-2133.