New rabies viral resources for multi-scale neural circuit mapping.

Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer's disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy.

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

Identification of long-lived proteins in the mitochondria reveals increased stability of the electron transport chain.

In order to combat molecular damage, most cellular proteins undergo rapid turnover. We have previously identified large nuclear protein assemblies that can persist for years in post-mitotic tissues and are subject to age-related decline. Here, we report that mitochondria can be long lived in the mouse brain and reveal that specific mitochondrial proteins have half-lives longer than the average proteome. These mitochondrial long-lived proteins (mitoLLPs) are core components of the electron transport chain (ETC) and display increased longevity in respiratory supercomplexes. We find that COX7C, a mitoLLP that forms a stable contact site between complexes I and IV, is required for complex IV and supercomplex assembly. Remarkably, even upon depletion of COX7C transcripts, ETC function is maintained for days, effectively uncoupling mitochondrial function from ongoing transcription of its mitoLLPs. Our results suggest that modulating protein longevity within the ETC is critical for mitochondrial proteome maintenance and the robustness of mitochondrial function.

Elemental mapping of labelled biological specimens at intermediate energy loss in an energy-filtered TEM acquired using a direct detection device.

The technique of colour EM that was recently developed enabled localisation of specific macromolecules/proteins of interest by the targeted deposition of diaminobenzidine (DAB) conjugated to lanthanide chelates. By acquiring lanthanide elemental maps by energy-filtered transmission electron microscopy (EFTEM) and overlaying them in pseudo-colour over the conventional greyscale TEM image, a colour EM image is generated. This provides a powerful tool for visualising subcellular component/s, by the ability to clearly distinguish them from the general staining of the endogenous cellular material. Previously, the lanthanide elemental maps were acquired at the high-loss M4,5 edge (excitation of 3d electrons), where the characteristic signal is extremely low and required considerably long exposures. In this paper, we explore the possibility of acquiring the elemental maps of lanthanides at their N4,5 edge (excitation of 4d electrons), which occurring at a much lower energy-loss regime, thereby contains significantly greater total characteristic signal owing to the higher inelastic scattering cross-sections at the N4,5 edge. Acquiring EFTEM lanthanide elemental maps at the N4,5 edge instead of the M4,5 edge, provides ∼4× increase in signal-to-noise and ∼2× increase in resolution. However, the interpretation of the lanthanide maps acquired at the N4,5 edge by the traditional 3-window method, is complicated due to the broad shape of the edge profile and the lower signal-above-background ratio. Most of these problems can be circumvented by the acquisition of elemental maps with the more sophisticated technique of EFTEM Spectrum Imaging (EFTEM SI). Here, we also report the chemical synthesis of novel second-generation DAB lanthanide metal chelate conjugates that contain 2 lanthanide ions per DAB molecule in comparison with 0.5 lanthanide ion per DAB in the first generation. Thereby, fourfold more Ln3+ per oxidised DAB would be deposited providing significant amplification of signal. This paper applies the colour EM technique at the intermediate-loss energy-loss regime to three different cellular targets, namely using mitochondrial matrix-directed APEX2, histone H2B-Nucleosome and EdU-DNA. All the examples shown in the paper are single colour EM images only.

Genetic Probe for Visualizing Glutamatergic Synapses and Vesicles by 3D Electron Microscopy.

Communication between neurons relies on the release of diverse neurotransmitters, which represent a key-defining feature of a neuron's chemical and functional identity. Neurotransmitters are packaged into vesicles by specific vesicular transporters. However, tools for labeling and imaging synapses and synaptic vesicles based on their neurochemical identity remain limited. We developed a genetically encoded probe to identify glutamatergic synaptic vesicles at the levels of both light and electron microscopy (EM) by fusing the mini singlet oxygen generator (miniSOG) probe to an intralumenal loop of the vesicular glutamate transporter-2. We then used a 3D imaging method, serial block-face scanning EM, combined with a deep learning approach for automatic segmentation of labeled synaptic vesicles to assess the subcellular distribution of transporter-defined vesicles at nanometer scale. These tools represent a new resource for accessing the subcellular structure and molecular machinery of neurotransmission and for transmitter-defined tracing of neuronal connectivity.

Split-miniSOG for Spatially Detecting Intracellular Protein-Protein Interactions by Correlated Light and Electron Microscopy.

A protein-fragment complementation assay (PCA) for detecting and localizing intracellular protein-protein interactions (PPIs) was built by bisection of miniSOG, a fluorescent flavoprotein derived from the light, oxygen, voltage (LOV)-2 domain of Arabidopsis phototropin. When brought together by interacting proteins, the fragments reconstitute a functional reporter that permits tagged protein complexes to be visualized by fluorescence light microscopy (LM), and then by standard as well as "multicolor" electron microscopy (EM) via the photooxidation of 3-3'-diaminobenzidine and its derivatives.

Age Mosaicism across Multiple Scales in Adult Tissues.

Most neurons are not replaced during an animal's lifetime. This nondividing state is characterized by extreme longevity and age-dependent decline of key regulatory proteins. To study the lifespans of cells and proteins in adult tissues, we combined isotope labeling of mice with a hybrid imaging method (MIMS-EM). Using 15N mapping, we show that liver and pancreas are composed of cells with vastly different ages, many as old as the animal. Strikingly, we also found that a subset of fibroblasts and endothelial cells, both known for their replicative potential, are characterized by the absence of cell division during adulthood. In addition, we show that the primary cilia of beta cells and neurons contains different structural regions with vastly different lifespans. Based on these results, we propose that age mosaicism across multiple scales is a fundamental principle of adult tissue, cell, and protein complex organization.

Visualization of long-lived proteins reveals age mosaicism within nuclei of postmitotic cells.

Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.

Iron-specific Signal Separation from within Heavy Metal Stained Biological Samples Using X-Ray Microtomography with Polychromatic Source and Energy-Integrating Detectors.

Biological samples are frequently stained with heavy metals in preparation for examining the macro, micro and ultra-structure using X-ray microtomography and electron microscopy. A single X-ray microtomography scan reveals detailed 3D structure based on staining density, yet it lacks both material composition and functional information. Using a commercially available polychromatic X-ray source, energy integrating detectors and a two-scan configuration labelled by their energy- "High" and "Low", we demonstrate how a specific element, here shown with iron, can be detected from a mixture with other heavy metals. With proper selection of scan configuration, achieving strong overlap of source characteristic emission lines and iron K-edge absorption, iron absorption was enhanced enabling K-edge imaging. Specifically, iron images were obtained by scatter plot material analysis, after selecting specific regions within scatter plots generated from the "High" and "Low" scans. Using this method, we identified iron rich regions associated with an iron staining reaction that marks the nodes of Ranvier along nerve axons within mouse spinal roots, also stained with osmium metal commonly used for electron microscopy.

Sub-mitochondrial localization of the genetic-tagged mitochondrial intermembrane space-bridging components Mic19, Mic60 and Sam50.

Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs.

Deceleration of probe beam by stage bias potential improves resolution of serial block-face scanning electron microscopic images.

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the  primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.

Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

The influence of the sample thickness on the lateral and axial resolution of aberration-corrected scanning transmission electron microscopy.

The lateral and axial resolution of three-dimensional (3D) focal series aberration-corrected scanning transmission electron microscopy was studied for samples of different thicknesses. The samples consisted of gold nanoparticles placed on the top and at the bottom of silicon nitride membranes of thickness between 50 and 500 nm. Atomic resolution was obtained for nanoparticles on top of 50-, 100-, and 200-nm-thick membranes with respect to the electron beam traveling downward. Atomic resolution was also achieved for nanoparticles placed below 50-, 100-, and 200-nm-thick membranes but with a lower contrast at the larger thicknesses. Beam broadening led to a reduced resolution for a 500-nm-thick membrane. The influence of the beam broadening on the axial resolution was also studied using Monte Carlo simulations with a 3D sample geometry.

The probe profile and lateral resolution of scanning transmission electron microscopy of thick specimens.

Lateral profiles of the electron probe of scanning transmission electron microscopy (STEM) were simulated at different vertical positions in a micrometers-thick carbon sample. The simulations were carried out using the Monte Carlo method in CASINO software. A model was developed to fit the probe profiles. The model consisted of the sum of a Gaussian function describing the central peak of the profile and two exponential decay functions describing the tail of the profile. Calculations were performed to investigate the fraction of unscattered electrons as a function of the vertical position of the probe in the sample. Line scans were also simulated over gold nanoparticles at the bottom of a carbon film to calculate the achievable resolution as a function of the sample thickness and the number of electrons. The resolution was shown to be noise limited for film thicknesses less than 1 µm. Probe broadening limited the resolution for thicker films. The validity of the simulation method was verified by comparing simulated data with experimental data. The simulation method can be used as quantitative method to predict STEM performance or to interpret STEM images of thick specimens.

Optimized deconvolution for maximum axial resolution in three-dimensional aberration-corrected scanning transmission electron microscopy.

Three-dimensional (3D) datasets were recorded of gold nanoparticles placed on both sides of silicon nitride membranes using focal series aberration-corrected scanning transmission electron microscopy (STEM). Deconvolution of the 3D datasets was applied to obtain the highest possible axial resolution. The deconvolution involved two different point spread functions, each calculated iteratively via blind deconvolution. Supporting membranes of different thicknesses were tested to study the effect of beam broadening on the deconvolution. It was found that several iterations of deconvolution was efficient in reducing the imaging noise. With an increasing number of iterations, the axial resolution was increased, and most of the structural information was preserved. Additional iterations improved the axial resolution by maximal a factor of 4 to 6, depending on the particular dataset, and up to 8 nm maximal, but also led to a reduction of the lateral size of the nanoparticles in the image. Thus, the deconvolution procedure optimized for the highest axial resolution is best suited for applications where one is interested in the 3D locations of nanoparticles only.

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.

Atomic-resolution scanning transmission electron microscopy through 50-nm-thick silicon nitride membranes.

Silicon nitride membranes can be used for windows of environmental chambers for in situ electron microscopy. We report that aberration corrected scanning transmission electron microscopy (STEM) achieved atomic resolution on gold nanoparticles placed on both sides of a 50-nm-thick silicon nitride membrane at 200 keV electron beam energy. Spatial frequencies of 1∕1.2 Å were visible for a beam semi-angle of 26.5 mrad. Imaging though a 100-nm-thick membrane was also tested. The achieved imaging contrast was evaluated using Monte Carlo simulations of the STEM imaging of a sample of with a representative geometry and composition.

Diffraction imaging in a He+ ion beam scanning transmission microscope.

The scanning helium ion microscope has been used in transmission mode to investigate both the feasibility of this approach and the utility of the signal content and the image information available. Operating at 40 keV the penetration of the ion beam, and the imaging resolution achieved, in MgO crystals was found to be in good agreement with values predicted by Monte Carlo modeling. The bright-field and annular dark-field signals displayed the anticipated contrasts associated with beam absorption and scattering. In addition, the diffraction of the He ion beam within the sample gave rise to crystallographic contrast effects in the form of thickness fringes and dislocation images. Scanning transmission He ion microscopy thus achieves useful sample penetration and provides nanometer scale resolution, high contrast images of crystalline materials and crystal defects even at modest beam energies.

A model of secondary electron imaging in the helium ion scanning microscope.

A combination of the 'semi-empirical' model for secondary electron production and the TRIM routines which describe ion stopping power, scattering, and transport, has been used to construct a Monte Carlo simulation (IONiSE) that can quantitatively interpret the generation of secondary electrons (SE) from materials by fast helium ions. This approach requires that the parameters of the semi-empirical model be determined by fitting to experimental yield data but has the merit that, unlike more fundamental models, it can be applied with equal ease to both pure elements and complex compounds. The application of the model to predict the topographic yield variation of helium generated SE as a function of energy and material, and to investigate the ratio between SE generated by incident and backscattered ions, is demonstrated.