The trend towards laboratory automation has resulted in an increasing demand for efficient quality control of cell cultures, driving the development of high-throughput solutions. A widely used method in quality control in cell cultivation is phase contrast microscopy, as it allows for the visualization of high-contrast images without requiring cell staining. However, its effectiveness is limited by the meniscus effect, which confines the visible region in cell culture vessels, such as microtiter plates (MTPs), to the small flat center of each well. Moreover, state-of-the-art phase contrast microscopy is time-consuming for large samples. This thesis addresses both limitations by exploring a rapid method to counteract the meniscus effect in well plates, enabling the acquisition of high-contrast composite images across entire wells. A meniscus effect compensation technique termed adaptive phase contrast microscopy is developed, which inserts adaptive elements into the microscope’s illumination path. Experiments revealed that replacing the fixed condenser annulus with a Liquid Crystal Display (LCD) yields the best results among adaptive elements, outperforming an adjustable liquid-filled prism. High-speed imaging is achieved through a continuous scanning technique that captures numerous microscopy images during sample movement, which are subsequently stitched into a large composite image. The LCD adaptively modifies the illumination for each image to compensate for the meniscus effect. To evaluate the phase contrast area within MTPs, the relative background intensity in images proved to be a reliable indicator of phase contrast conditions. Experimental results demonstrate that adaptive phase contrast microscopy significantly enhances the phase contrast area. The phase contrast area increased up to ninefold, depending on the MTP type and magnification used. Key factors influencing the phase contrast area include the magnification and numerical aperture of the objective lens, shading from the well walls, image field curvature, and total internal reflection at steep surface angles. Notably, the rapid acquisition speed, matching the LCD’s refresh rate of 60 Hz, did not compromise image quality. Furthermore, experiments demonstrated that imaging durations could be effectively reduced to be only slightly slower than those achieved by high-speed microscopy for conventional phase contrast microscopy, and up to 25 times faster than traditional stop-and-go methods. Crucial factors affecting acquisition speed include the LCD’s refresh rate and latency, the necessity to limit acceleration to prevent liquid sloshing, and the range of the microscope stage. In conclusion, it was demonstrated that the phase contrast area in MTPs can be significantly enhanced through meniscus effect compensation, and high-speed imaging is feasible without degrading image quality.
Florian Nienhaus (Thu,) studied this question.