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We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with

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

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.

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 > 106 µm3. 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. DOI: http://dx.doi.org/10.7554/eLife.25916.001, eLife digest Precise three-dimensional imaging can help make sense of microscopic details in biology. These images are usually built up from many two-dimensional images stacked on top of each other. One approach for examining particularly fine details, such as the connections between nerve cells in the brain, is called focused ion beam scanning electron microscopy (or FIB-SEM for short). This approach works by creating an image of the surface layer of a sample, which is then stripped away using a beam of charged particles to reveal the layer beneath. The new surface can then be imaged and so on, through the whole sample. Unfortunately, FIB-SEM devices are currently slow and can only run for a short time, leading to a lack of continuity in the stack of images. FIB-SEM would allow faster, more accurate and detailed studies of connections between brain cells, and other elaborate biological systems, if the technology could be made faster and more reliable over months of continuous operation. The current technical challenge is to create a system that can, for example, successfully image and analyse all the connections between the more than 100 thousand cells that make up the brain of a fruit fly – a common model organism in neurobiology. Xu et al. aimed to create a technique to image a complete fly brain, with gaps of just 8 nanometres between each image in a stack, within a reasonable timeframe. By improving how FIB-SEM signals are detected, making use of advances in ion beam controls, and by engineering ways to recover from system malfunctions, Xu et al. developed an enhanced FIB-SEM device. To demonstrate its value, the new technology was used to create images of a third of a fruit fly’s brain, parts of a mouse’s brain, and cells of a single-celled alga called Chlamydomonas reinhardtii. The results show that large and complex samples can be successfully imaged in their entirety to adequate detail, enabling high-quality reconstruction of the connections between nerve cells. The level of detail, which can be further increased for smaller samples, offers advantages in precision and image quality over other comparable techniques. As well as helping to study the brain, this approach could also be used to examine details inside cells. Future work to advance this technology will enable larger and more complete imaging of elaborate biological structures. DOI: http://dx.doi.org/10.7554/eLife.25916.002

We report the first application of three-dimensional (3D) single-particle tracking (SPT) to hydrodynamic size characterization of gold nanoparticles in water. Nanoparticles undergoing Brownian motion were dynamically locked at the focal point of a microscope objective, one at a time, by rapid counteractive movements of the sample container. The hydrodynamic radius was derived from the recorded trajectory of each individual nanoparticle. The directly measured size and size distribution using 3D-SPT were in agreement with those obtained using the conventional dynamic light scattering (DLS) and using transmission electron microscopy (TEM), respectively.

In this paper we discuss an end-point detection (EPD) method for the dielectric linear chemical–mechanical planarization (CMP) processes. The proposed EPD algorithms utilize the interferometry optical signals to determine the post-CMP film thickness. A set of collected broadband spectral signals are formed as a spectral image. An image-matching technique is then used to match the processed signal image to the reference image template obtained at the target film thickness. Several matching criteria are discussed and compared. We find that the image correlation coefficient is a good indicator to determine the process end-point. We also consider the impact of the material removal rate variations on the interferometry spectral signals. An analytical calculation is carried out to find an extraction and compression searching range of the spectral image to compensate for the removal rate uncertainties. The correctness and effectiveness of the proposed algorithms have been demonstrated through applications to an inter-metal dielectric (IMD) device CMP process. Compared with other optical EPD methods, the proposed image-matching method is robust to the CMP process variations.

Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm x 10 nm x 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 mu m thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then 'volume stitched' back together, producing a final three-dimensional data set suitable for connectome tracing.

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.

In this paper we discuss an end-point detection (EPD) method for the dielectric linear chemical-mechanical planarization (CMP) processes. The proposed EPD algorithms utilize the interferometry optical signals to determine the film post-thicknesses. A set of collected broadband spectral signals is formed as an spectral image. An image-matching technique is then used to match the pre-processed signal image to the reference image template obtained at the target film thickness. Several matching criteria are discussed and compared. We find that the image correlation coefficient is a good indicator to determine the process end-point. We also consider the impact of the material removal rate variations on the interferometry spectral signals. An analytical calculation is carried out to find an extraction and compression searching range of the spectral image to compensate for the removal rate uncertainties in real processes. The correctness and effectiveness of the proposed algorithms have been demonstrated through applications to an inter-metal dielectric (IMD) device CMP process. Compared with other optical EPD methods, the proposed image-matching method are robust to the CMP process variations.

We present an apparatus that noninvasively tracks a moving nanoparticle in three dimensions while providing concurrent sequential spectroscopic measurements. The design, based on confocal microscopy, uses a near-infrared laser and a dark-field condenser for illumination of a gold nanoparticle. By monitoring the scattered light from the nanoparticle and using a piezoelectric stage, the system was able to continuously bring the diffusive particle in a glycerol/water solution back to the focal volume with spatial resolution and response time of less than 210nm and a millisecond, respectively.

Chemical mechanical planarization (CMP) is an enabling technology in the fabrication of advanced semiconductor devices. The surface topography of the starting bare silicon substrate before CMP can have a significant impact on the results of the CMP process, specifically on the uniformity of the oxide film final thickness. The quantitative studies of this phenomenon are just beginning. In this article, a laser based optical scanning measurement was used to determine the front surface topography of the bare silicon substrates prior to thermal oxide growth. Fast two-dimensional mapping of the surface height variation is obtained with very high spatial and height resolution. The one-dimensional cross-section profile extracted from the measurement is demonstrated to have good correlation with the stylus based profiler measurement. A thermal oxide layer was grown and then polished in a high efficiency planarization process on a group of substrates, which had different levels of surface topography variations on ...