Compensating the Meniscus Effect in Phase Contrast Microscopy Using an LCD for Adaptive Condenser Annulus Shifting

The meniscus effect in cell culture vessels limits the observable areas with phase contrast microscopy. For meniscus effect compensation in microtiter plates (MTPs), we present a method using an LCD to replace the fixed condenser annulus, which enables adaptive annulus shifting based on image analysis. This approach led to an increase in phase contrast area by a factor of 8.3. Utilizing a standard phase contrast microscope, we substituted the static condenser annulus with a transparent LCD that displays an adaptive annulus, which can be repositioned to counteract meniscus-induced refraction across an entire MTP-24 well. We developed image analysis using Bertrand lens images to determine the misalignment between annulus center and phase ring, enabling the calculation of the required annulus shift. Experiments demonstrate the effectiveness of this image analysis technique. The detected shift was translated into new LCD settings through a linear regression model to ensure proper alignment for the following image. We proved that an algorithm based on background brightness yields a reliable metric for assessing phase contrast conditions within well-plates. The proposed approach substantially increased the phase contrast area in 24-well MTPs at 10× magnification from 5.0% with conventional microscopy to 41.9%, thereby restoring phase contrast conditions throughout the well, except near the edges.