Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure

Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H(2)O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits

A Dendrite-Autonomous Mechanism for Direction Selectivity in Retinal Starburst Amacrine Cells

Detection of image motion direction begins in the retina, with starburst amacrine cells (SACs) playing a major role. SACs generate larger dendritic Ca2+ signals when motion is from their somata towards their dendritic tips than for motion in the opposite direction. To study the mechanisms underlying the computation of direction selectivity (DS) in SAC dendrites, electrical responses to expanding and contracting circular wave visual stimuli were measured via somatic whole-cell recordings and quantified using Fourier analysis. Fundamental and, especially, harmonic frequency components were larger for expanding stimuli. This DS persists in the presence of GABA and glycine receptor antagonists, suggesting that inhibitory network interactions are not essential. The presence of harmonics indicates nonlinearity, which, as the relationship between harmonic amplitudes and holding potential indicates, is likely due to the activation of voltage-gated channels. [Ca2+] changes in SAC dendrites evoked by voltage steps and monitored by two-photon microscopy suggest that the distal dendrite is tonically depolarized relative to the soma, due in part to resting currents mediated by tonic glutamatergic synaptic input, and that high-voltage–activated Ca2+ channels are active at rest. Supported by compartmental modeling, we conclude that dendritic DS in SACs can be computed by the dendrites themselves, relying on voltage-gated channels and a dendritic voltage gradient, which provides the spatial asymmetry necessary for direction discrimination

Alpha-Herpesvirus Infection Induces the Formation of Nuclear Actin Filaments

Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process

Automated Segmentation of Large 3D Images of Nervous Systems Using a Higher-order Graphical Model

This thesis presents a new mathematical model for segmenting volume images. The model is an energy function defined on the state space of all possibilities to remove or preserve splitting faces from an initial over-segmentation of the 3D image into supervoxels. It decomposes into potential functions that are learned automatically from a small amount of empirical training data. The learning is based on features of the distribution of gray values in the volume image and on features of the geometry and topology of the supervoxel segmentation. To be able to extract these features from large 3D images that consist of several billion voxels, a new algorithm is presented that constructs a suitable representation of the geometry and topology of volume segmentations in a block-wise fashion, in log-linear runtime (in the number of voxels) and in parallel, using only a prescribed amount of memory. At the core of this thesis is the optimization problem of finding, for a learned energy function, a segmentation with minimal energy. This optimization problem is difficult because the energy function consists of 3rd and 4th order potential functions that are not submodular. For sufficiently small problems with 10,000 degrees of freedom, it can be solved to global optimality using Mixed Integer Linear Programming. For larger models with 10,000,000 degrees of freedom, an approximate optimizer is proposed and compared to state-of-the-art alternatives. Using these new techniques and a unified data structure for multi-variate data and functions, a complete processing chain for segmenting large volume images, from the restoration of the raw volume image to the visualization of the final segmentation, has been implemented in C++. Results are shown for an application in neuroscience, namely the segmentation of a part of the inner plexiform layer of rabbit retina in a volume image of 2048 x 1792 x 2048 voxels that was acquired by means of Serial Block Face Scanning Electron Microscopy (Denk and Horstmann, 2004) with a resolution of 22nm x 22nm x 30nm. The quality of the automated segmentation as well as the improvement over a simpler model that does not take geometric context into account, are confirmed by a quantitative comparison with the gold standard

Inhibitory Phenotype of HBV-Specific CD4⁺ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background: T-cell exhaustion seems to play a critical role in CD8(+) T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4(+) T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4(+) T-cell failure. Methods: The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4(+) T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4(+) T-cell proliferation and cytokine production. Results: CD4(+) T-cell responses during chronic HBV infection was characterized by reduced Tetramer(+)CD4(+) T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-gamma, IL-2 and TNF-alpha secretion as well as enhanced CD4(+) T-cell expansion almost in treated patients with viral control. Conclusion: HBV-specific CD4(+) T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4(+) T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4(+) T-cell functionality with heterogeneous patterns of CD4(+) T-cell rejunivation

Functional Fluorescent Ca(2+) Indicator Proteins in Transgenic Mice under TET Control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2

High speed optical coherence microscopy with autofocus adjustment and a miniaturized endoscopic imaging probe

Optical coherence microscopy (OCM) is a promising technique for high resolution cellular imaging in human tissues. An OCM system for high-speed en face cellular resolution imaging was developed at 1060 nm wavelength at frame rates up to 5 Hz with resolutions of < 4 µm axial and < 2 µm transverse. The system utilized a novel polarization compensation method to combat wavelength dependent source polarization and achieve broadband electro-optic phase modulation compatible with ultrahigh axial resolution. In addition, the system incorporated an auto-focusing feature that enables precise, near real-time alignment of the confocal and coherence gates in tissue, allowing user-friendly optimization of image quality during the imaging procedure. Ex vivo cellular images of human esophagus, colon, and cervix as well as in vivo results from human skin are presented. Finally, the system design is demonstrated with a miniaturized piezoelectric fiber-scanning probe which can be adapted for laparoscopic and endoscopic imaging applications.National Institutes of Health (U.S.) (R01-CA75289-13)National Institutes of Health (U.S.) R01-EY11289-25United States. Air Force Office of Scientific Research (FA9550-07-1-0101)United States. Air Force Office of Scientific Research (FA9550-07-1-0014)Max Planck Society for the Advancement of ScienceNational Institutes of Health (U.S.) (Fellowship) (F31 EB005978

Dysfunctional CD8(+) T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction

The transcription factor T-bet regulates the production of interferon-γ and cytotoxic molecules in effector CD8 T cells, and its expression correlates with improved control of chronic viral infections. However, the role of T-bet in infections with differential outcome remains poorly defined. Here, we report that high expression of T-bet in virus-specific CD8 T cells during acute hepatitis B virus (HBV) and hepatitis C virus (HCV) infection was associated with spontaneous resolution, whereas T-bet deficiency was more characteristic of chronic evolving infection. T-bet strongly correlated with interferon-γ production and proliferation of virus-specific CD8 T cells, and its induction by antigen and IL-2 stimulation partially restored functionality in previously dysfunctional T-bet–deficient CD8 T cells. However, restoration of a strong interferon-γ response required additional stimulation with IL-12, which selectively induced the phosphorylation of STAT4 in T-bet(+) CD8 T cells. The observation that T-bet expression rendered CD8 T cells responsive to IL-12 suggests a stepwise mechanism of T cell activation in which T-bet facilitates the recruitment of additional transcription factors in the presence of key cytokines. These findings support a critical role of T-bet for viral clearance and suggest T-bet deficiency as an important mechanism behind chronic infection