High-speed phase-contrast microscopy with adaptive meniscus effect compensation

We introduce a high-speed method for compensating the meniscus effect in phase-contrast microscopy that increases the area observable under phase-contrast conditions substantially while enabling rapid whole–microtiter-plate imaging. The meniscus effect in liquid-filled wells restricts phase-contrast imaging to a central region, here termed the phase-contrast area. Multiple compensation methods have been proposed, but an increased phase contrast area requires a high-throughput imaging process to limit total scan time. Our approach meets this need by combining meniscus effect compensation with high-speed scanning in a two-step workflow consisting of one scan to precompute condenser annulus positions and a second scan for imaging the specimen. Compensation is achieved by inserting a transparent LCD into the illumination path to display an adaptive virtual condenser annulus as a binary pattern. A continuous scanning mode enables high-speed imaging, in which shots are acquired during sample motion. The LCD is synchronized with the sample movement and image acquisition using path-synchronous triggers, with latency compensation for the LCD. Experiments show that the phase-contrast area increases by up to 8.96-fold relative to standard phase-contrast microscopy, depending on well size and magnification. We validated our approach against a slower and less complex stop-and-go method, which yielded similar phase-contrast areas, indicating no adverse effect of continuous acquisition on image quality. In contrast, the total scan time was reduced by a factor of up to 24.6 compared with stop-and-go, from 439 min to 17 min 52 s for 96-well MTPs at 10 (Formula presented) (Formula presented) magnification, achieved at frame rates of up to 60 frames per second, demonstrating the effectiveness of the proposed method.