Simultaneous Immersion Mirau Interferometry

The present work describes a novel imaging technique for label-free no-UV vibration-insensitive imaging of live cells in an epi-illumination geometry. This technique can be implemented in a variety of imaging applications. For example, it can be used for cell targeting as a part of a platform for targeted cell irradiations - single-cell microbeam. The goal of microbeam facilities is to provide biological researchers with tools to study the effects of ionizing radiation on live cells. A common way of cell labeling - fluorescent staining - may alter cellular metabolism and UV illumination presents potential damage for the genetic material. The new imaging technique will allow the researchers to separate radiation-induced effects from the effects caused by confounding factors like fluorescent staining or UV light. Geometry of irradiation endstations at some microbeam facilities precludes the use of transmitted light, e.g. in the Columbia University's Radiological Research Accelerator Facility microbeam endstation, where the ion beam exit window is located just below the sample. Imaging techniques used at such endstations must use epi-illumination. Mirau Interferometry is an epi-illumination, non-stain imaging modality suitable for implementation at a microbeam endstation. To facilitate interferometry and to maintain cell viability, it is desirable that cells stay in cell growth medium during the course of an experiment. To accommodate the use of medium, Immersion Mirau Interferometry has been developed. A custom attachment for a microscope objective has been designed and built for interferometric imaging with the possibility of immersion of the apparatus into cell medium. The implemented data collection algorithm is based on the principles of Phase-Shifting Interferometry. The largest limitation of Phase-Shifting Interferometry is its sensitivity to the vertical position of the sample. In environments where vibration isolation is difficult, this makes image acquisition challenging. This problem was resolved by integration of polarization optics into the optics of the attachment to enable simultaneous creation and spatial separation of two interferograms, which, combined with the background image, are used to reconstruct the intensity map of the specimen. Giving the name Simultaneous Immersion Mirau Interferometry to this approach, simultaneous acquisition of all interferograms per image has eliminated the issue of vibrations. The designed compound microscope attachment has been manufactured and tested; the system produces images of quality, sufficient to perform targeted cellular irradiation experiments.