Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-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 entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. 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.

In conventional biological imaging, diffraction places a limit on the minimal xy distance at which two marked objects can be discerned. Consequently, resolution of target molecules within cells is typically coarser by two orders of magnitude than the molecular scale at which the proteins are spatially distributed. Photoactivated localization microscopy (PALM) optically resolves selected subsets of protect fluorescent probes within cells at mean separations of <25 nanometers. It involves serial photoactivation and subsequent photobleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this paper, some of the limitations for PALM imaging under current experimental conditions are discussed.

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

A central problem in neuroscience is reconstructing neuronal circuits on the synapse level. Due to a wide range of scales in brain architecture such reconstruction requires imaging that is both high-resolution and high-throughput. Existing electron microscopy (EM) techniques possess required resolution in the lateral plane and either high-throughput or high depth resolution but not both. Here, we exploit recent advances in unsupervised learning and signal processing to obtain high depth-resolution EM images computationally without sacrificing throughput. First, we show that the brain tissue can be represented as a sparse linear combination of localized basis functions that are learned using high-resolution datasets. We then develop compressive sensing-inspired techniques that can reconstruct the brain tissue from very few (typically 5) tomographic views of each section. This enables tracing of neuronal processes and, hence, high throughput reconstruction of neural circuits on the level of individual synapses.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 10(6) µm(3). These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.

A gas of hydrogen atoms, confined in a static magnetic trap, has been evaporatively cooled to temperatures of a few millikelvin. The initial trap configuration held the gas at 38 mK for as long as 5 h. Evaporative cooling reduced the temperature to 3.0 mK while maintaining the central density at 7.6×10 12   cm −3   . These values were determined by measurement of the rate of electronic spin relaxation and are in agreement with model calculations. Further cooling to 1 mK (inferred from the model) has been achieved. Measurements were made of the efficiency of the evaporative cooling process.