Total internal reflection holographic microcopy for cell extension imaging

Abstract

The study of interfacial structures is of utmost importance not only for various research fields such as cell biology and display systems but also their sub-disciplines. One of the traditional means of imaging buried structures rely on fluorescence labeling and the use of optical sectioning with superresolution microscopy. Although it exceeds diffraction limit, there are various shortcomings to utilize this methodology such as its reliance on fluorescent markers, long exposure times to high cost of the imaging sys tem. Ultimately, these limitations position the existing technologies unideal for live cell imaging, including the imaging of surface proteins of a living cell. A label free quanti tative phase imaging method is realized in this study to enable imaging of an interface between different media. This system is based on off-axis holographic microscopy and uses a high numerical aperture (NA) microscope objective to achieve total internal reflection (TIR). Existing literature on total internal reflection holographic microscopy utilizes prism to achieve TIR which limits the working distance of objective to be large hence resolution. Proposed system relies on a 100x objective with 1.49 NA to improve resolution and magnification. Complex field which is reflected from burried interface of the sample can be recovered by using digital holography principles. The resolution of the system can further be enhanced by combining several illumination angles and utilizing synthetic aperture reconstruction. Also a new iterative algorithm for maskless grayscale lithography which uses quantitative phase measurements as feedback is real ized in this study. Phase measurements are performed via an off-axis digital holography configuration. A spiral phase plate which has uses in superresolution microscopy tech niques is produced with proposed algorithm and shown to perform better compared to classical method.