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Light sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.

Lattice light sheet microscopy excels at the non-invasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here we undertake a comprehensive theoretical and experimental analysis of various forms of light sheet microscopy which both demonstrates and explains why lattice light sheets provide significant improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. Development of a new type of “harmonic balanced” lattice light sheet is shown to improve performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.Significance StatementDespite its rapidly growing use, several misconceptions remain concerning the physics of image formation and its optimization in light sheet microscopy, particularly in high resolution variants tailored for subcellular imaging. These include the role of excitation sidelobes, the significance of out-of-focus fluorescence, the importance and optimization of deconvolution, and the perceived advantages of Gaussian beams. Here we attempt to shatter these misconceptions by showing that the professed tradeoffs between axial resolution and background haze, photobleaching rate, phototoxicity, and propensity for image artifacts do not exist for well-crafted lattice light sheets whose data is acquired and processed rigorously. The framework we provide should enable others to optimize light sheets and extract the most information at the lowest cost in their experiments.

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at unprecedented depths in vivo.

Visualizing whole cells at many scales Cells need to compartmentalize thousands of distinct proteins, but the nanoscale spatial relationship of many proteins to overall intracellular ultrastructure remains poorly understood. Correlated light and electron microscopy approaches can help. Hoffman et al. combined cryogenic super-resolution fluorescence microscopy and focused ion beam–milling scanning electron microscopy to visualize protein-ultrastructure relationships in three dimensions across whole cells. The fusion of the two imaging modalities enabled identification and three-dimensional segmentation of morphologically complex structures within the crowded intracellular environment. The researchers observed unexpected relationships within a variety of cell types, including a web-like protein adhesion network between juxtaposed cerebellar granule neurons. Science , this issue p. eaaz5357

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

INTRODUCTION Neural circuits across the brain are composed of structures spanning seven orders of magnitude in size that are assembled from thousands of distinct protein types. Electron microscopy has imaged densely labeled brain tissue at nanometer-level resolution over near-millimeter-level dimensions but lacks the contrast to distinguish specific proteins and the speed to readily image multiple specimens. Conversely, confocal fluorescence microscopy offers molecular contrast but has insufficient resolution for dense neural tracing or the precise localization of specific molecular players within submicrometer-sized structures. Last, superresolution fluorescence microscopy bleaches fluorophores too quickly for large-volume imaging and also lacks the speed for effective brain-wide or cortex-wide imaging of multiple specimens. RATIONALE We combined two imaging technologies to address these issues. Expansion microscopy (ExM) creates an expanded, optically clear phantom of a fluorescent specimen that retains its original relative distribution of fluorescent tags. Lattice light-sheet microscopy (LLSM) then images this phantom in three dimensions with minimal photobleaching at speeds sufficient to image the entire Drosophila brain or across the width of the mouse cortex in ∼2 to 3 days, with multiple markers at an effective resolution of ∼60 by 60 by 90 nm for 4× expansion. RESULTS We applied expansion/LLSM (ExLLSM) to study a variety of subcellular structures in the brain. In the mouse cortex, we quantified the volume of organelles, measured morphological parameters of ~1500 dendritic spines, determined the variation of distances between pre- and postsynaptic proteins, observed large differences in postsynaptic expression at adjacent pyramidal neurons, and studied both the azimuthal asymmetry and layer-specific longitudinal variation of axonal myelination. In Drosophila , we traced the axonal branches of olfactory projection neurons across one hemisphere and studied the stereotypy of their boutons at the calyx and lateral horn across five animals. We also imaged all dopaminergic neurons (DANs) across the brain of another specimen, visualized DAN morphologies in all major brain regions, and traced a cluster of eight DANs to their termini to determine their respective cell types. In the same specimen, we also determined the number of presynaptic active zones (AZs) across the brain and the local density of all AZs and DAN-associated AZs in each brain region. CONCLUSION With its high speed, nanometric resolution, and ability to leverage genetically targeted, cell type–specific, and protein-specific fluorescence labeling, ExLLSM fills a valuable niche between the high throughput of conventional optical pipelines of neural anatomy and the ultrahigh resolution of corresponding EM pipelines. Assuming the development of fully validated, brain-wide isotropic expansion at 10× or beyond and sufficiently dense labeling, ExLLSM may enable brainwide comparisons of even densely innervated neural circuits across multiple specimens with protein-specific contrast at 25-nm resolution or better.

Summary Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. Video Abstract, Graphical Abstract, Highlights • A complete adult Drosophila brain was imaged with EM and has been made publicly available • The imaged volume enables brain-spanning mapping of circuits at synaptic resolution • All mushroom body (MB) calyx inputs were mapped, revealing a new cell type, MB-CP2 • Previously unidentified synaptic partners form recurrent microcircuits in MB calyx, Electron microscopy imaging of the entire adult fruit fly brain at synapse resolution reveals circuitry spanning multiple regions and connectivity between known and previously unknown cell types.

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entireDrosophilabrain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.One Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.

In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.One Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

Wavefront-shaping devices incorporated into optical microscopy systems are capable of correcting sample-induced aberrations and recovering diffraction-limited imaging performance. The widespread dissemination and application of adaptive optical techniques, however, requires easy integration of adaptive optical modules, both in terms of hardware and software, into existing microscopes. We built an adaptive optical module with reduced complexity and simplified integration by utilizing a novel segmented deformable mirror and a standalone control software program. We demonstrated its ability to improve image brightness and resolution at depth in the mouse, zebrafish, and fly brains in vivo.

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

Adding to the super-resolution arsenal Structured illumination microscopy (SIM) uses light intensities that are orders of magnitude lower than other super-resolution methods. SIM is also far faster over cellular-sized fields of view. Li et al. used two approaches to improve the resolution of SIM to allow live cell imaging of dynamic cellular processes, including endocytosis and cytoskeleton remodeling. The contrast in performance between SIM and other techniques is due to a few key differences. Defining the practical resolution at the limited signal-to-noise ratios necessary for live cell imaging will require better imaging metrics. Science , this issue 10.1126/science.aab3500