Quantitative Analysis of Biological Cells Using Digital Holographic Microscopy

Abstract

Biological cells are microscopic dynamic objects, continuously adjusting their threedimensional sizes, shapes and other biophysical features. Wide-field microscopy of cell dynamics can provide a powerful research tool for cell biology studies, as well as a potential means for medical diagnosis and monitoring of diseases. Biological cells, however, are mostly-transparent objects, and thus imaging them with conventional intensity-based light microscopy fails to provide adequate optical contrast between the cell and its environment. Although exogenous contrast agents such as fluorescent dyes can be used to solve this problem, they might be cytotoxic in the long run and there is a possibility they will influence the cellular behavior. Additionally, fluorescent dyes tend to photobleach, potentially limiting the imaging time. The contrast problem when imaging biological cells can also be solved by using phase microscopy, which records the optical path delays of light passing through the cells and subsequently obtains information on the cellular structure and dynamics without using any exogenous labelling. Since detectors are sensitive to intensity only, the phase of the light that has interacted with the cells must first be converted to intensity variations for detection. Widely used methods to achieve this include phase contrast microscopy and differential interference contrast (DIC) microscopy. However, these techniques are not inherently quantitative and present distinct imaging artifacts that typically prevent straightforward extraction of the entire optical path delay profile of the cell. Wide-field digital interferometry (WFDI) is a label-free holographic technique that is able to record the entire complex wavefront (amplitude and phase) of the light which has interacted with the sample (Cuche et al., 1999). From the recorded complex field, one can obtain full quantitative phase profiles of cells as well as correct for out-of-focus image features by postprocessing. WFDI microscopy (also called digital holographic microscopy) has been applied to various types of biological cell systems and has recorded a diverse range of cellular phenomena (Marquet et al., 2005; Ikeda et al., 2005; Shaked et al., 2009 b; Shaked et al., 2010 b-d). Section 2 reviews the principle of WFDI for obtaining phase profiles of cells, starting from the experimental setup, and ending in digital processing for obtaining the final quantitative phase profile of the cell. Although WFDI is a quantitative recording technique, simple quasi-three-dimensional holographic visualization of the cell phase profile need not be the end of the process.