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Extracellular vesicles (EVs) are plasma-membrane formed particles released by cells, and range in diameter from 50 to 2000 nm. Interest in EVs is growing, and recent work has aimed to employ nonlinear optical microscopy techniques to better characterize the size, function, and biochemical makeup of EVs. Previous studies have shown that EVs can modulate gene expression and metabolism of cells that uptake them. Here, we use fluorescence lifetime imaging microscopy (FLIM) of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) to monitor the metabolic response of macrophages and other cells to native and foreign EVs and other known cellular activators.

Multimodal imaging systems are designed to extract complementary multifaceted information from biological samples; sensorless adaptive optics (AO) seeks to extend their capabilities by estimating the wavefront from image-derived information. Typical sensorless AO techniques need the acquisition of several volumes for optimization. We present a novel single-shot closed-loop sensorless AO technique demonstrated on a label-free multimodal imaging system consisting of optical coherence, two-photon fast fluorescence lifetime imaging, and second harmonic generation microscopy. The wavefront is sensed by performing computational AO on an initial OCM volume that is translated to the deformable mirror to improve the image quality of all modalities in real-time.