Fluorescence microscopy shadow imaging for neuroscience.

Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membrane-bound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility.

The Impact of Chemical Fixation on the Microanatomy of Mouse Organotypic Hippocampal Slices.

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy (EM). However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles, and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts, and knowing how to avoid them, takes on special urgency. We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a "before-and-after" experiment. We applied super-resolution shadow imaging (SUSHI) to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters. While the ECS volume fraction (VF) and microanatomic organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted. Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphologic preservation of cells and tissues.

Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge.

The extracellular space (ECS) plays a central role in brain physiology, shaping the time course and spread of neurochemicals, ions, and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain's parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS. However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with unprecedented spatial resolution in a live experimental setting. Focusing on ring-like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical interface between astrocytic and cellular structures. Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil, and the ECS in living brain tissue, which could be of functional relevance for neuron-glia communication in a variety of (patho)physiological settings, for example, LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.

Author Correction: Structural basis of astrocytic Ca2+ signals at tripartite synapses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis of astrocytic Ca2+ signals at tripartite synapses.

Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.

Salivary gland macrophages and tissue-resident CD8+ T cells cooperate for homeostatic organ surveillance.

It is well established that tissue macrophages and tissue-resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. In contrast, the mechanisms by which these two cell types cooperate for homeostatic organ surveillance after clearance of infections is poorly understood. Here, we used intravital imaging to show that TRM dynamically followed tissue macrophage topology in noninflamed murine submandibular salivary glands (SMGs). Depletion of tissue macrophages interfered with SMG TRM motility and caused a reduction of interepithelial T cell crossing. In the absence of macrophages, SMG TRM failed to cluster in response to local inflammatory chemokines. A detailed analysis of the SMG microarchitecture uncovered discontinuous attachment of tissue macrophages to neighboring epithelial cells, with occasional macrophage protrusions bridging adjacent acini and ducts. When dissecting the molecular mechanisms that drive homeostatic SMG TRM motility, we found that these cells exhibit a wide range of migration modes: In addition to chemokine- and adhesion receptor-driven motility, resting SMG TRM displayed a remarkable capacity for autonomous motility in the absence of chemoattractants and adhesive ligands. Autonomous SMG TRM motility was mediated by friction and insertion of protrusions into gaps offered by the surrounding microenvironment. In sum, SMG TRM display a unique continuum of migration modes, which are supported in vivo by tissue macrophages to allow homeostatic patrolling of the complex SMG architecture.

A super-resolution platform for correlative live single-molecule imaging and STED microscopy.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.

Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue.

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

Multifunctional plasmonic film for recording near-field optical intensity.

We demonstrate the plasmonic equivalent of photographic film for recording optical intensity in the near field. The plasmonic structure is based on gold bowtie nanoantenna arrays fabricated on SiO2 pillars. We show that it can be employed for direct laser writing of image data or recording the polarization structure of optical vector beams. Scanning electron micrographs reveal a careful sculpting of the radius of curvature and height of the triangles composing the illuminated nanoantennas, as a result of plasmonic heating, that permits spatial tunability of the resonance response of the nanoantennas without sacrificing their geometric integrity. In contrast to other memory-dedicated approaches using Au nanorods embedded in a matrix medium, plasmonic film can be used in multiple application domains. To demonstrate this functionality, we utilize the structures as plasmonic optical tweezers and show sequestering of SiO2 microparticles into optically written channels formed between exposed sections of the film. The plasmonic film offers interesting possibilities for photonic applications including optofluidic channels "without walls," in situ tailorable biochemical sensing assays, and near-field particle manipulation and sorting.

Application of quantitative second-harmonic generation microscopy to dynamic conditions.

We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

Rotational Doppler-effect due to selective excitation of vector-vortex field in optical fiber.

Experimental demonstration of rotational Doppler-effect due to direct and simultaneous excitation of orthogonal elliptically-polarized fundamental and vortex modes in a two-mode optical fiber is presented here. The rotation frequency and the trajectory of the zero-intensity point in the two-mode fiber output beam measured as a function of analyzer rotation matches with the S-contour of polarization singularity in the beam, identified via Stokes parameter measurement. The characteristics of the S-contour around the C-point in the output beam is also measured as a function of rotating Dove prism and half-wave plate - Dove prism combination to highlight the role of polarization modifying components on the observed rotational Doppler-effect of vector-vortex beams.