Label-free Rheinberg staining of cells using digital holographic microscopy and spatial light interference microscopy

The problem that is posed by "phase-only" objects, such as epithelial cells, for brightfield microscopy has resulted in development of several specialized imaging techniques including phase-contrast and DIC. In the past decades there has been increasing research on quantitative phase imaging (QPI), which enables real-time cellular dynamics to be visualised and the 3-D morphology to be quantitatively measured. It has previously been demonstrated that the DIC and phase-contrast images can be computationally generated using the phase-image provided by QPI. Recently, we have extended this approach to include Rheinberg. Although not as popular as phase contrast or DIC, Rheinberg illumination provides a form of label-free optical staining by introducing a multi-color filter into the condenser plane of the microscope, enabling different features within the cell to be stained with different colors depending on their spatial-frequency content. We recently developed a theory for image formation with Rheinberg illumination under the conditions of Kohler illumination from which an algorithm was developed that could simulate this process using the QPI image as input. In this paper we review and further develop this approach by testing it with multiple different modalities for recording the QPI image, namely digital holographic microscopy, which uses coherent illumination and spatial light interference microscopy, which makes use of white light. We examine a variety of samples including diatom and epithelial cells using a number of microscope objectives with different numerical apertures.