Adaptive illumination for optimal image quality in phase contrast microscopy

Abstract

Zernike phase contrast microscopy is a powerful and well established microscopy technique. Due to its ability to enhance contrast of transparent, unlabelled specimens, it is widely used for live cell imaging. But for samples contained in small culture dishes or multiwell plates filled with buffer solution, the curved surface of the liquid buffer distorts the illumination light path. This leads to a misalignment of the ring shaped illumination with respect to the phase ring inside the objective lens. As a consequence, the image contrast and quality can be severely degraded, especially when imaging at the margin of the containers.Here we present a solution to this problem that is based on using a programmable illumination unit. This allows us to compensate the illumination for the distortions induced by the interface meniscus. We find that a simple strategy, which is based on correcting illumination angles where possible and removing light in angles where such a correction is not possible, can maintain the image contrast over a large percentage of a culture dish bottom area.Furthermore, we show that our approach can be extended beyond the ring-shaped mask design of Zernike imaging to phase masks consisting of multiple spots, a method which has been shown to be superior regarding the minimization of artefacts and for quantitative phase measurements.