Engineering efficient intravital imaging to better eavesdrop on bology

Our two-photon light-sheet microscopy approach combines better engineering of the detection objective's point-spread-function, with optimized computational tools for hyperspectral and fluorescence lifetime imaging, to study living specimens. Our goal is to animate the wealth of high-throughput molecular data to better understand complex events, ranging from embryonic development to disease processes. This two-photon SPIM approach generates images with more than a10-fold improved imaging speed & sensitivity than conventional light sheet microscopes, resulting in 4D (3D over time) cell and molecular images that offer sufficient speed and resolution to unambiguous trace cell lineages, movements and signals in intact systems. Our new multispectral image analysis tools exceed the performance of our previous work by orders of magnitude in speed, error propagation and accuracy. Novel denoising strategies for hyperspectral and FLIM datasets, some using machine learning, permit imaging at far lower light levels, yielding rapid and unambiguous analyses without perturbing even fragile multiplex-labeled specimens.