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

Journal of Physics Photonics, vol. 8, pp. 025038

Abstract

Abstract 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 × magnification, achieved at frame rates of up to 60 frames per second, demonstrating the effectiveness of the proposed method.

Authors 4

1. Florian Nienhaus corresponding Aachen Fraunhofer Institute for Production Technology IPT

   Fraunhofer Institute for Production Technology IPT

   Affiliation as printed

   Fraunhofer Institute for Production Technology IPT

2. Caroline Girmen Aachen Fraunhofer Institute for Production Technology IPT

   Fraunhofer Institute for Production Technology IPT

   Affiliation as printed

   Fraunhofer Institute for Production Technology IPT

3. Niels Koenig Aachen Fraunhofer Institute for Production Technology IPT

   Fraunhofer Institute for Production Technology IPT

   Affiliation as printed

   Fraunhofer Institute for Production Technology IPT

4. Robert H. Schmitt Aachen Fraunhofer Institute for Production Technology IPT

   Fraunhofer Institute for Production Technology IPT · RWTH Aachen University

   Affiliation as printed

   Fraunhofer Institute for Production Technology IPT

   WZL | RWTH Aachen University