Single-objective selective-volume illumination microscopy enables high-contrast light-field imaging.

The performance of light-field microscopy is improved by selectively illuminating the relevant subvolume of the specimen with a second objective lens. Here we advance this approach to a single-objective geometry, using an oblique one-photon illumination path or two-photon illumination to accomplish selective-volume excitation. The elimination of the second orthogonally oriented objective to selectively excite the volume of interest simplifies specimen mounting; yet, this single-objective approach still reduces the out-of-volume background, resulting in improvements in image contrast, effective resolution, and volume reconstruction quality. We validate our new, to the best of our knowledge, approach through imaging live developing zebrafish, demonstrating the technology's ability to capture imaging data from large volumes synchronously with high contrast while remaining compatible with standard microscope sample mounting.

High-contrast, synchronous volumetric imaging with selective volume illumination microscopy.

Light-field fluorescence microscopy uniquely provides fast, synchronous volumetric imaging by capturing an extended volume in one snapshot, but often suffers from low contrast due to the background signal generated by its wide-field illumination strategy. We implemented light-field-based selective volume illumination microscopy (SVIM), where illumination is confined to only the volume of interest, removing the background generated from the extraneous sample volume, and dramatically enhancing the image contrast. We demonstrate the capabilities of SVIM by capturing cellular-resolution 3D movies of flowing bacteria in seawater as they colonize their squid symbiotic partner, as well as of the beating heart and brain-wide neural activity in larval zebrafish. These applications demonstrate the breadth of imaging applications that we envision SVIM will enable, in capturing tissue-scale 3D dynamic biological systems at single-cell resolution, fast volumetric rates, and high contrast to reveal the underlying biology.

Optimal wavelength combinations for near-infrared spectroscopic monitoring of changes in brain tissue hemoglobin and cytochrome c oxidase concentrations.

We analyze broadband near-infrared spectroscopic measurements obtained from newborn piglets subjected to hypoxia-ischemia and we aim to identify optimal wavelength combinations for monitoring cerebral tissue chromophores. We implement an optimization routine based on the genetic algorithm to perform a heuristic search for discrete wavelength combinations that can provide accurate concentration information when benchmarked against the gold standard of 121 wavelengths. The results indicate that it is possible to significantly reduce the number of measurement wavelengths used in conjunction with spectroscopic algorithms and still achieve a high performance in estimating changes in concentrations of oxyhemoglobin, deoxyhemoglobin, and oxidized cytochrome c oxidase. While the use of a 3-wavelength combination leads to mean recovery errors of up to 10%, these errors drop to less than 4% with 4 or 5 wavelengths and to even less than 2% with 8 wavelengths.