Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

SummaryDrosophila 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

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ~0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

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 a-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

Aerogels are ultralight, highly porous materials typically fabricated by subjecting a wet-gel precursor to critical-pointdrying (CPD) or lyophilization (freeze-drying) in order to remove background liquid without collapsing the network. Microscopically, aerogels are composed of tenuous networks of clustered nanoparticles, and the materials often have unique properties, including very high strength-to-weight and surface-area-to-volume ratios. To date most aerogels are fabricated from silica [1] or pyrolized organic polymers. [2,3] Practical interest in the former stems from their potential for ultralight structural media, radiation detectors, and thermal insulators, [1] and in the latter from their potential for battery electrodes and supercapacitors. [2] In this paper we investigate the properties of a new class of aerogels based on carbon nanotubes (CNTs). Small-diameter CNTs, such as single-and few-wall CNTs, are exciting candidates for electrically conducting aerogels. Individually, these nanotubes are extraordinarily stiff [4] and their electrical conductivity can be very large. [4,5] Furthermore, ensembles of such nanotubes are useful aerogel precursors: they form electrically percolating networks at very low volume fractions [6] and elastic gels in concentrated suspensions through van der Waals interaction mediated cross-linking. [7,8] Here we report the creation of CNT aerogels from aqueousgel precursors by CPD and freeze-drying. CNT aerogels have been produced previously as intermediate phases during the process of drawing nanotube fibers [9] from a furnace and during the process of making sheets from multiwall CNT forests.[10] By contrast, our aerogels were derived directly from CNT networks in suspension, and we could readily manipulate the network properties as a result. The flexibility afforded by this process enabled us to control CNT concentration, to utilize optimized CNT dispersion processes, [11] to reinforce the networks with, for example, polyvinyl alcohol (PVA), and to infiltrate or backfill them with polymeric fluids. Here we describe these CNT aerogels and the processing methodologies used to synthesize them, and we characterize their electrical and mechanical properties. The CNT aerogels supported thousands of times their own weight after PVA-reinforcement, and, depending on processing conditions, their electrical conductivity ranged as high as ca. 1 S cm -1. Although our starting chemical vapor deposition (CVD) nanotube material contained single-and few-wall CNTs (the latter being predominantly double-wall CNTs, DWNTs), the dispersion and preparation processes employed here are directly applicable to pure single-wall CNTs (SWNTs).[11] CNT aerogel electrical and structural properties are also expected to be similar to pure SWNT samples because the electrical [12] and tensile [13] properties of bulk SWNTs and DWNTs are comparable. Images of typical critical-point-dried aerogels are seen in Figure 1. Unreinforced aerogels were fragile, but strong enough to permit careful handling. Reinforceme...

Graphical Abstract Highlights d Super-resolution live-cell imaging up to 266 fps at 97-nm resolution d Hitchhiking interactions among organelles remodel ER and mitochondrial networks d ER-mitochondrion contacts promote coalescence of mitochondrial membranes d Collision of late endosomes or lysosomes carried along microtubules split ER tubules

Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction-limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 microm.

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 that 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 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.