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Innovations in each modality improved the overall speed and sensitivity. Fast fluorescence lifetime imaging microscopy (FLIM) was accelerated with our computational photon counting algorithm called single-and-multi photon peak event detection (SPEED), capable of counting up to 250% photon rates. Dual-channel fast two-photon FLIM was achieved by compressed sensing of analog photocurrents on a field-programmable gate array on board the digitizer. We developed a new and faster method for hyperspectral coherent Raman microscopy using supercontinuum generation and custom pulse shapers, which facilitated rapid tuning to desired vibrational states. Polarization multiplexing in optical coherence imaging enabled compressed sensing of birefringence. Finally, computational approaches to maximize the information from these complementary contrasts yielded new insights into the processes within the tissue microenvironment. VAMPIRE microscopy is the nexus of label-free microscopy research, advances in optoelectronic technologies, and our innovations in computational and multimodal imaging for diverse applications.
Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle θmin that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of θmin. At a setting of θmin = 20 deg, the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As θmin is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at θmin = 20 deg for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.
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