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.

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.

Analysis and isolation of cell cultures play a decisive role in biological processes. Currently, these steps are often performed manually, which is time-consuming. Additionally, for each step a separate device is required. The aim of LIFTOSCOPE is to automate these processes in just one device. It combines three functional principles: high-speed microscopy, image analysis and laser-induced forward transfer. A prototype was successfully developed to demonstrate the feasibility of this concept.

Bei der bereits veröffentlichten Originalversion des Artikels wurde die Angabe der Bildquelle in Abbildung 1 vergessen. Diese ist: [11] Narrog F, Lensing R, Piotrowski T et al. (2023) LIFTOSCOPE: development of an automated AI-based module for time-effective and contactless analysis and isolation of cells in microtiter plates. J Biol Eng 17: 10. https://doi.org/10.1186/s13036-023-00329-9. © Fraunhofer ILT.