DEVELOPMENT OF A MULTIDIMENSIONAL FLUORESCENCE MICROSCOPE USING MULTIPOINT CONFOCAL SCANNING

Abstract

Capturing cellular dynamics is key to understanding cell behavior, but this task is challenging due to the weak fluorescence signal in live cells. This signal scarcity becomes more pronounced when divided across multiple contrast dimensions, pushing the boundaries of detector sensitivity. This complexity of measurement is essential for revealing the intricate mechanisms governing cellular function. By using spatial, spectral, and fluorescence lifetime imaging contrasts, we can more precisely isolate species and interactions, uncovering previously hidden aspects of cellular behavior. In this work, we present the development of multiple prototypes for multi-dimensional multipoint confocal microscopy, designed to optimize the use of these faint signals and advance the study of cellular dynamics. Our prototype systems, unmatched in speed and spectral resolution, utilize a pinhole array for efficient confocal multiplexing and dense time-resolved detectors, such as a gated optical intensifier, to measure multipoint confocal time-resolved fluorescence spectra. We demonstrate an enhanced optical design using a 32x32 pinhole array and a SPAD array to capture 960x960 pixel images at a frame rate of 4 Hz. Additionally, we present a 10x10 point multispectral FLIM system, representing the first highly multiplexed multispectral confocal FLIM microscope. A novel optical design further improves the acquisition rate by reducing the sensor readout rate requirements from a quadratic sampling problem to a linear sampling problem. This new optical system can capture 22 spectral bands simultaneously across the 450 nm to 650 nm spectral range at a 1Hz frame rate with a final image resolution of 960x1920. These advancements mark a significant step towards realizing a high-speed multipoint multispectral confocal FLIM microscope and lay the groundwork for future improvements and research.