Application of modular digital holographic microscopy to clinical pathology samples before histopathological staining

Digital Holography microscopy is a widely applied technique in biological imaging for retrieving quantitative phase information relating to a samples three-dimensional morphology and has received considerable interest in the investigation of real-time cellular dynamics. However, the traditional off-axis architecture has drawbacks in terms of a requirement for frequent calibration when the sample is changed as a consequence of the optical path length variation. This inconvenience can be overcome using a common-path design microscopy. Here we report on a portable module utilizing an off-axis architecture and an inexpensive laser diode source. The portability of the system enables it to be added to easily to the output port of an existing life-science microscope. An important feature of the module is that it is based on the self-reference principle, whereby the reference wavefield is generated from the image wavefield that is exiting the microscope image port using a suitable beam splitter and filter. This facilitates the source laser to be completely decoupled from the module. We investigate the application of the module to image samples of cells prepared using a common pathology slide preparation method known as ThinPrep, which is often used to prepare ovarian cancer, urine/bladder, and oral cytology samples for diagnostic inspection and to provide quantitative morphological information. In particular, we are interested in imaging samples that are fixed and air dried such that no mounting medium or coverslip is present, with the motivation of recording phase images immediately prior to cell staining and mounting.