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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) . In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca 2+ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals. Single molecule fluorescence microscopy is a powerful technique to study protein dynamics in cells, but it has not been applied to adult animals. The authors use complementation-activated light microscopy in C. elegans to discover that dystrophin regulates the diffusion properties of voltage-dependent calcium ion channels at the surface of body-wall muscle cells.

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. Thai Truong et al. present light-field-based selective volume illumination microscopy (SVIM), a method for enhancing image contrast and resolution by combining light-field microscopy and selective plane illumination microscopy. They generate cellular-resolution 3D movies by applying SVIM to flowing bacteria in seawater and to the beating heart and whole brain of larval zebrafish.

High-numerical-aperture (NA) oblique plane microscopy enables noninvasive fluorescence imaging of subcellular dynamics without requiring radical sample modification. However, performance degrades at depth in multicellular specimens as scattering and refractive-index heterogeneity raise out-of-focus background. We report a two-photon oblique plane microscope that improves resolution at depth by combining high-NA single-objective detection with multiphoton plane illumination. The microscope achieves $\\sim\\!300$ nm lateral and $\\sim\\!650$ nm axial resolution, with single-molecule sensitivity in vivo. Compared with two-photon point scanning, the lower illumination NA delivers an order of magnitude lower peak intensity, enabling $>\\!5\\times$ faster volumetric acquisition (up to $3.25 \\times 10^6$ voxels s$^{-1}$) with reduced photodamage. In multicellular contexts, near-infrared nonlinear excitation enhances contrast throughout the illumination depth by $\\sim\\!2\\times$ and restores volumetric resolving power by $>\\!2\\times$ relative to linear excitation. We demonstrate these capabilities through molecular imaging of epithelial tissue, stem-cell-derived gastruloids, and living fruit fly embryos, including multicolor transcription-factor dynamics, optogenetic subcellular control, and single-mRNA tracking, all using standard glass-based mounting.

Light-sheet microscopes must compromise between field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide high resolution but their narrow depth of field fails to capture effectively the fluorescence signal generated by the illumination light sheets, in 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. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases total fluorescence collection, reduces background, and improves the overall signal-to-noise ratio (SNR). We demonstrate, through numerical simulations and imaging of bead phantoms as well as living animals, that ExD-SPIM increases the SNR by more than three-fold and dramatically reduces the rate of fluorescence photobleaching, when compared to a low-NA system with an equivalent depth of field. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.

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. We used machine learning to predict the stimulus from the neuronal activity and observed that the prediction accuracy improves with age. We further tested ethanol's effect on number sense and found a decrease in number-selective neurons in the forebrain, suggesting cognitive impairment. These findings are a significant step towards understanding neural circuits devoted to discrete magnitudes and our methodology to track single-neuron activity across the whole brain is broadly applicable to other fields in neuroscience.

The performance of light-field microscopy is improved by selectively illuminating the relevant subvolume of the specimen with a second objective lens [1-3]. 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 out-of-volume background, resulting in improvements in image contrast, effective resolution, and volume reconstruction quality. We validate our new 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.

Live biotherapeutic products (LBPs) are emerging as powerful tools to modulate the microbiome using well-defined microbial communities. Yet, designing, manufacturing, and delivering LBPs remains challenging, in part due to a lack of technologies capable of analyzing LBPs as complete, spatially organized consortia. Conventional sequencing-based methods lack sensitivity and specificity and do not provide critical spatial information. To address this, we present high-phylogenetic-resolution spatial mapping platform (HiPR-Map), a state-of-the-art spectral imaging technology that enables precise enumeration and spatial localization of microbial cells at species-level within complex communities. Through these advantages, HiPR-Map provides unique insights for LBP discovery and development. Applying HiPR-Map to an LBP designed to complement immune checkpoint therapy, we profiled over 1.8 million microbial cells engrafted in the murine gut. Our analysis revealed distinctive microbial spatial organization, underscoring the power of imaging-based microbiome profiling to optimize LBP design and characterization. This work highlights the transformative potential of spatial microbiome analysis for next-generation LBP development.

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 an instrument, the flex-SPIM, that combines two independently controlled light-sheet microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing throughput and decreasing cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright and/or inverted detection geometry. We offer a detailed description of the flex-SPIM design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument's versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.