Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy

Label-free quantitative in vitro imaging of living cell cultures with light microscopy is an important tool for various research fields in the life sciences. Digital holographic microscopy (DHM) provides contactless, minimally invasive quantitative phase contrast imaging and can be integrated as a module in common research microscopes. Due to the numerical reconstruction of quantitative phase images, multi-focus imaging is achieved from a single digital hologram. The evaluation of the recorded quantitative phase contrast images allows the extraction of data for simplified object tracking and image segmentation. The special DHM feature of numerical autofocusing avoids mechanical focus realignment. As quantitative DHM phase imaging is based on the detection of optical path length changes in transmission, the method only requires low light intensities for object illumination which minimizes the interaction with the sample. Thus, minimally invasive long-term time-lapse investigations for quantitative monitoring of dynamic changes of cell morphology, motility, and proliferation are accessible. In addition, the integral cellular refractive index, which is related to intracellular solute concentrations as well as cellular volume and dry mass, is available. The chapter starts with an introduction to DHM for live cell observation and procedures for the extraction of biophysical parameters from quantitative DHM phase contrast images. After the physical basis has been laid out, several selected applications of in vitro live cell analysis are described. This includes the characterization of suspended cells and spherical intracellular organelles as well as the quantification of the cellular response to osmotic stimulation, drugs, toxins, nanomaterials, and genetic modifications. Subsequent paragraphs illustrate how DHM can be applied to quantify cell motility, migration, and the morphology of adherent cell cultures. Finally, phenotyping based on cell thickness determination, dynamic multimodal imaging of cellular growth, proliferation, and wound healing in vitro as well as applications in toxicity testing of pathogens and the characterization of cell nanomaterial interactions are demonstrated.

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