Neural Basis of Number Sense in Larval Zebrafish.

Number sense, the ability to discriminate the quantity of objects, is crucial for survival. To understand how neurons work together and develop to mediate number sense, we used two-photon fluorescence light sheet microscopy to capture the activity of individual neurons throughout the brain of larval , while displaying a visual number stimulus to the animal. We identified number-selective neurons as early as 3 days post-fertilization and found a proportional increase of neurons tuned to larger quantities after 3 days.

A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy.

Hyperspectral fluorescence imaging improves multiplexed observations of biological samples by utilizing multiple color channels across the spectral range to compensate for spectral overlap between labels. Typically, spectral resolution comes at a cost of decreased detection efficiency, which both hampers imaging speed and increases photo-toxicity to the samples. Here, we present a high-speed, high-efficiency snapshot spectral acquisition method, based on optical compression of the fluorescence spectra via Fourier transform, that overcomes the challenges of discrete spectral sampling: single-shot hyperspectral phasor camera (SHy-Cam).

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture.

Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet.

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.

A versatile, multi-laser twin-microscope system for light-sheet imaging.

Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here, we present a versatile light-sheet imaging instrument that combines two independently controlled microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing the throughput and decreasing the cost.

Circulating Tumor Cells: High-Throughput Imaging of CTCs and Bioinformatic Analysis.

Circulating tumor cells (CTCs) represent novel biomarkers, since they are obtainable through a simple and noninvasive blood draw or liquid biopsy. Here, we review the high-definition single-cell analysis (HD-SCA) workflow, which brings together modern methods of immunofluorescence with more sophisticated image processing to rapidly and accurately detect rare tumor cells among the milieu of platelets, erythrocytes, and leukocytes in the peripheral blood. In particular, we discuss progress in methods to measure CTC morphology and subcellular protein expression, and we highlight some initial applications that lead to fundamental new insights about the hematogenous phase of cancer, as well as its performance in early-stage diagnosis and treatment monitoring.

Paired High-Content Analysis of Prostate Cancer Cells in Bone Marrow and Blood Characterizes Increased Androgen Receptor Expression in Tumor Cell Clusters.

Recent studies demonstrate that prostate cancer clones from different metastatic sites are dynamically represented in the blood of patients over time, suggesting that the paired evaluation of tumor cells in circulation and bone marrow, the primary target for prostate cancer metastasis, may provide complementary information. We adapted our single-cell high-content liquid biopsy platform to bone marrow aspirates (BMA) to concurrently identify and characterize prostate cancer cells in patients' blood and bone and thus discern features associated to tumorigenicity and dynamics of metastatic progression. The incidence of tumor cells in BMAs increased as the disease advanced: 0% in biochemically recurrent ( = 52), 26% in newly diagnosed metastatic hormone-naïve ( = 26), and 39% in metastatic castration-resistant prostate cancer (mCRPC; = 63) patients, and their number was often higher than in paired blood.

In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans.

Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans).