Quantitative phase imaging for label‐free cytometry

INTRODUCTION IN order to understand complex biological processes, scientists must gain insights into the functioning of individual live cells. Unlike fixed cell imaging, where a single snapshot of the cell’s life is retrieved, live-cell imaging allows investigation of the dynamic processes underlying the cell’s function. Labelfree imaging avoids the limitations inherent to fluorescent probes (phototoxicity, photobleaching) and maintains an appropriate environment for normal cellular behavior. Typical mammalian cells are transparent phase objects which do not absorb or scatter much light. Enhanced visualization of unlabeled cells was enabled in 1932 by the development of phase contrast (PC) technique by Frits Zernike (1,2) who, during the decade that followed, collaborated with Carl Zeiss AG resulting in a release of the first phase contrast microscope, revolutionizing the field of live cell dynamic imaging. PC is based on an optical set-up that translates small variations in phase into corresponding changes in amplitude, which consequently can be visualized as differences in image contrast. PC and various derivatives, such as differential interference contrast, became widely adopted techniques for imaging thin live cells in culture. However, these methods only provide a means for visualizing the cells and not making measurements, that is, the phase information they output is qualitative. Traditional PC techniques are not quantitative as they do not provide a direct measurement of a phase delay with enumeration as pixel intensity. This special issue is focused on introducing to our readership the subject of Quantitative Phase Imaging (QPI) (3) and its benefits to cytometry. QPI is a valuable method for studying live cell dynamics, as it provides a noninvasive analysis over a wide range of time scales. This type of analysis is gaining traction very rapidly because it is performed with little to no phototoxicity and requires minimal sample preparation. There are no effects of biological and chemical labels or genetic modification, which would alter cellular behavior. QPI offers the benefit of repeated observations and quantitative analysis of cell cultures over time providing minute-byminute insight into cell proliferation, cell death, and transient events. Quantitative measurements are based on direct phase image analysis of cell structure. QPI yields optical path difference maps associated with the specimen of interest and, as such, it is sensitive to both local thickness and the refractive index of the sample. Several QPI related publications have previously appeared in Cytometry Part A, paving the way for this new field of applications (4–6). A collection of manuscripts in this special issue attests to the fact that QPI is becoming a prominent technique complementary to traditional cytometry technologies and indispensable in dynamic label-free live-cell analysis applications.