Short infrared laser pulses (SILP) have many physiological effects on cells, including the ability to stimulate action potentials (APs) in neurons. Here, we show that SILPs can also reversibly block APs. Reversible AP block in hippocampal neurons was observed following SILP (0.26 to 0.96  J/cm²; 1.37 to 5.01 ms; 1869 nm) with the block persisting for more than 1 s with exposures greater than 0.69  J/cm². AP block was sustained for 30 s with SILPs pulsed at 1 to 7 Hz. Full recovery of neuronal activity was observed 5 to 30 s post SILP exposure. These results indicate that SILP can be used for noncontact, reversible AP block. Due to the high spatial precision and noncontact manner of infrared light delivery, AP block by SILP (infrared neural inhibition) has the potential to transform medical care for sustained pain inhibition and suppression of unwanted nerve activity.

This PDF file contains the editorial “Special Section Guest Editorial: Super-resolution microscopy of neural structure and function” for NPh Vol. 3 Issue 04

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap—acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Advancement in fluorescence imaging with the invention of several super-resolution microscopy modalities (e.g., PALM/STORM and STED) has opened up the possibility of deciphering molecular distributions on the nanoscale. In our quest to better elucidate postsynaptic protein distribution in dendritic spines, we have applied these nanoscopy methods, where generated results could help improve our understanding of neuronal functions. In particular, we have investigated the principal energy transformer in the brain, i.e., the Na+,K+-ATPase (or sodium pump), an essential protein responsible for maintaining resting membrane potential and a major controller of intracellular ion homeostasis. In these investigations, we have focused on estimates of protein amount, giving assessments of how variations may depend on labeling strategies, sample analysis, and choice of nanoscopic imaging method, concluding that all can be critical factors for quantification. We present a comparison of these results and discuss the influences this may have for homeostatic sodium regulation in neurons and energy consumption.

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The caliber of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. We quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (D_mean) in neurites from 0.12±0.05 to 0.61±0.03  μm²/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9±1.9 to 15±4.9  μm/s, whereas a velocity increase from 9±1.3 to 14±3  μm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

The ability to count molecules is essential to elucidating cellular mechanisms, as these often depend on the absolute numbers and concentrations of molecules within specific compartments. Such is the case at chemical synapses, where the transmission of information from presynaptic to postsynaptic terminals requires complex interactions between small sets of molecules. Be it the subunit stoichiometry specifying neurotransmitter receptor properties, the copy numbers of scaffold proteins setting the limit of receptor accumulation at synapses, or protein packing densities shaping the molecular organization and plasticity of the postsynaptic density, all of these depend on exact quantities of components. A variety of proteomic, electrophysiological, and quantitative imaging techniques have yielded insights into the molecular composition of synaptic complexes. In this review, we compare the different quantitative approaches and consider the potential of single molecule imaging techniques for the quantification of synaptic components. We also discuss specific neurobiological data to contextualize the obtained numbers and to explain how they aid our understanding of synaptic structure and function.

Emerging all-optical methods provide unique possibilities for noninvasive studies of physiological processes at the cellular and subcellular scale. On the one hand, superresolution microscopy enables observation of living samples with nanometer resolution. On the other hand, light can be used to stimulate cells due to the advent of optogenetics and photolyzable neurotransmitters. To exploit the full potential of optical stimulation, light must be delivered to specific cells or even parts of cells such as dendritic spines. This can be achieved with computer generated holography (CGH), which shapes light to arbitrary patterns by phase-only modulation. We demonstrate here in detail how CGH can be incorporated into a stimulated emission depletion (STED) microscope for photostimulation of neurons and monitoring of nanoscale morphological changes. We implement an original optical system to allow simultaneous holographic photostimulation and superresolution STED imaging. We present how synapses can be clearly visualized in live cells using membrane stains either with lipophilic organic dyes or with fluorescent proteins. We demonstrate the capabilities of this microscope to precisely monitor morphological changes of dendritic spines after stimulation. These all-optical methods for cell stimulation and monitoring are expected to spread to various fields of biological research in neuroscience and beyond.

The misfolding and self-assembly of intrinsically disordered proteins into insoluble amyloid structures are central to many neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Optical imaging of this self-assembly process in vitro and in cells is revolutionizing our understanding of the molecular mechanisms behind these devastating conditions. In contrast to conventional biophysical methods, optical imaging and, in particular, optical superresolution imaging, permits the dynamic investigation of the molecular self-assembly process in vitro and in cells, at molecular-level resolution. In this article, current state-of-the-art imaging methods are reviewed and discussed in the context of research into neurodegeneration.

Recent developments in single-molecule imaging have revealed many biological mechanisms, providing high spatial and temporal resolution maps of molecular events. In neurobiology, these techniques unveiled that plasma membrane neurotransmitter receptors and transporters laterally diffuse at the surface of cultured brain cells. The photostability of bright nanoprobes, such as quantum dots (QDs), has given access to neurotransmitter receptor tracking over long periods of time with a high spatial resolution. However, our knowledge has been restricted to cultured systems, i.e., neurons and organotypic slices, therefore lacking several aspects of the intact brain rheology and connectivity. Here, we used QDs to track single glutamatergic N-methyl-d-aspartate receptors (NMDAR) in acute brain slices. By delivering functionalized nanoparticles in vivo through intraventricular injections to rats expressing genetically engineered-tagged NMDAR, we successfully tracked the receptors in native brain tissue. Comparing NMDAR tracking to different classical brain preparations (acute brain slices, cultured organotypic brain slices, and cultured neurons) revealed that the surface diffusion properties shared several features and are also influenced by the nature of the extracellular environment. Together, we describe the experimental procedures to track plasma membrane NMDAR in dissociated and native brain tissue, paving the way for investigations aiming at characterizing receptor diffusion biophysics in intact tissue and exploring the physiopathological roles of receptor surface dynamics.

High voltage gated calcium channels (VGCCs) are composed of at least three subunits, one pore forming α₁-subunit, an intracellular β-variant, and a mostly extracellular α₂δ-variant. Interactions between these subunits determine the kinetic properties of VGCCs. It is unclear whether these interactions are stable over time or rather transient. Here, we used single-molecule tracking to investigate the surface diffusion of α₁- and α₂δ₁-subunits at the cell surface. We found that α₂δ₁-subunits show higher surface mobility than α₁-subunits, and that they are only transiently confined together, suggesting a weak association between α₁- and α₂δ₁-subunits. Moreover, we observed that different α₁-subunits engage in different degrees of association with the α₂δ₁-subunit, revealing the tighter interaction of α₂δ₁ with Ca_V1.2<Ca_V2.2<Ca_V2.1<Ca_V3.2. These data indicate a distinct regulation of the α₁/α₂δ₁ interaction in VGCC subtypes. We modeled their membrane dynamics in a Monte Carlo simulation using experimentally determined diffusion constants. Our modeling predicts that the ratio of associated α₁- and α₂δ₁-subunits mainly depends on their expression density and confinement in the membrane. Based on the different motilities of particular α₁/α₂δ₁-subunit combinations, we propose that their dynamic assembly and disassembly represent an important mechanism to regulate the signaling properties of VGCC.

The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-1β (Nrx1β), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin. The small probe (∼3  nm) efficiently penetrates into crowded synaptic junctions and reduces the distance to target. We quantified the organization of the single-molecule localization data using a tesselation-based analysis technique. We show that Nlg1 exhibits a fairly disperse organization within dendritic spines, while LRRTM2 is organized in compact domains, and Nrx1β in presynaptic terminals displays a dual-organization pattern intermediate between that of Nlg1 and LRRTM2. These results suggest that part of Nrx1β interacts transsynaptically with Nlg1 and the other part with LRRTM2

Receptor trafficking and its regulation have appeared in the last two decades to be a major controller of basal synaptic transmission and its activity-dependent plasticity. More recently, considerable advances in super-resolution microscopy have begun deciphering the subdiffraction organization of synaptic elements and their functional roles. In particular, the dynamic nanoscale organization of neurotransmitter receptors in the postsynaptic membrane has recently been suggested to play a major role in various aspects of synapstic function. We here review the recent advances in our understanding of alpha-amino-3-hydroxy-5-méthyl-4-isoxazolepropionic acid subtype glutamate receptors subsynaptic organization and their role in short- and long-term synaptic plasticity.

Monitoring the changes of cerebral hemodynamics and the state of consciousness during general anesthesia (GA) is clinically important. There is a great need for developing advanced detectors to investigate the physiological processes of the brain during GA. We developed a multichanneled, functional near-infrared spectroscopy (fNIRS) system device and applied it to GA operation monitoring. The cerebral hemodynamic data from the forehead of 11 patients undergoing propofol and sevoflurane anesthesia were analyzed. The concentration changes of oxygenated hemoglobin, deoxygenated hemoglobin, total hemoglobin, and cerebral tissue heart rate were determined from the raw optical information based on the discrete stationary wavelet transform. This custom-made device provides an easy-to-build solution for continuous wave-fNIRS system, with customized specifications. The developed device has a potential value in cerebral monitoring in clinical settings.

Atlas-guided diffuse optical tomography (atlas-DOT) is a computational means to image changes in cortical hemodynamic signals during human brain activities. Graph theory analysis (GTA) is a network analysis tool commonly used in functional neuroimaging to study brain networks. Atlas-DOT has not been analyzed with GTA to derive large-scale brain connectivity/networks based on near-infrared spectroscopy (NIRS) measurements. We introduced an automated voxel classification (AVC) method that facilitated the use of GTA with atlas-DOT images by grouping unequal-sized finite element voxels into anatomically meaningful regions of interest within the human brain. The overall approach included volume segmentation, AVC, and cross-correlation. To demonstrate the usefulness of AVC, we applied reproducibility analysis to resting-state functional connectivity measurements conducted from 15 young adults in a two-week period. We also quantified and compared changes in several brain network metrics between young and older adults, which were in agreement with those reported by a previous positron emission tomography study. Overall, this study demonstrated that AVC is a useful means for facilitating integration or combination of atlas-DOT with GTA and thus for quantifying NIRS-based, voxel-wise resting-state functional brain networks.

Using light, we are able to visualize the hemodynamic behavior of the brain to better understand neurovascular coupling and cerebral metabolism. In vivo optical imaging of tissue using endogenous chromophores necessitates spectroscopic detection to ensure molecular specificity as well as sufficiently high imaging speed and signal-to-noise ratio, to allow dynamic physiological changes to be captured, isolated, and used as surrogate of pathophysiological processes. An optical imaging system is introduced using a 16-bands on-chip hyperspectral camera. Using this system, we show that up to three dyes can be imaged and quantified in a tissue phantom at video-rate through the optics of a surgical microscope. In vivo human patient data are presented demonstrating brain hemodynamic response can be measured intraoperatively with molecular specificity at high speed.

We report the development of a compact probe for time-domain (TD) functional near-infrared spectroscopy (fNIRS) based on a fast silicon photomultiplier (SiPM) that can be put directly in contact with the sample without the need of optical fibers for light collection. We directly integrated an avalanche signal amplification stage close to the SiPM, thus reducing the size of the detection channel and optimizing the signal immunity to electromagnetic interferences. The whole detection electronics was placed in a plastic screw holder compatible with the electroencephalography standard cap for measurement on brain or with custom probe holders. The SiPM is inserted into a transparent and insulating resin to avoid the direct contact of the scalp with the 100-V bias voltage. The probe was integrated in an instrument for TD fNIRS spectroscopy. The system was characterized on tissue phantoms in terms of temporal resolution, responsivity, linearity, and capability to detect deep absorption changes. Preliminary in vivo tests on adult volunteers were performed to monitor hemodynamic changes in the arm during a cuff occlusion and in the brain cortex during a motor task.

The cerebral metabolic rate of oxygen (CMRO₂) is an essential parameter for evaluating brain function and pathophysiology. However, the currently available approaches for quantifying CMRO₂ rely on complex multimodal imaging and mathematical modeling. Here, we introduce a method that allows estimation of CMRO₂ based on a single measurement modality—two-photon imaging of the partial pressure of oxygen (PO₂) in cortical tissue. We employed two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen-sensitive nanoprobe PtP-C343 to map the tissue PO₂ distribution around cortical penetrating arterioles. CMRO₂ is subsequently estimated by fitting the changes of tissue PO₂ around arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO₂ in anesthetized rats and modulated tissue PO₂ levels by manipulating the depth of anesthesia. This method provides CMRO₂ measurements localized within ∼200  μm and it may provide oxygen consumption measurements in individual cortical layers or within confined cortical regions, such as in ischemic penumbra and the foci of functional activation.

Enabling simultaneous high-resolution imaging of the total concentration of hemoglobin (C_Hb), oxygen saturation of hemoglobin (sO₂), and cerebral blood flow (CBF), multiparametric photoacoustic microscopy (PAM) holds the potential to quantify the cerebral metabolic rate of oxygen at the microscopic level. However, its imaging speed has been severely limited by the pulse repetition rate of the dual-wavelength photoacoustic excitation and the scanning mechanism. To address these limitations, we have developed a new generation of multiparametric PAM. Capitalizing on a self-developed high-repetition dual-wavelength pulsed laser and an optical–mechanical hybrid-scan configuration, this innovative technique has achieved an unprecedented A-line rate of 300 kHz, leading to a 20-fold increase in the imaging speed over our previously reported multiparametric PAM that is based on pure mechanical scanning. The performance of the high-speed multiparametric PAM has been examined both in vitro and in vivo. Simultaneous PAM of microvascular C_Hb, sO₂, and CBF in absolute values over a ∼3-mm-diameter brain region of interest can be accomplished within 10 min.

Optogenetics is one of the most important techniques in neurophysiology, with potential clinical applications. However, the strong light needed may cause harmful temperature rises. So far, there are no methods to reliably estimate brain heating and safe limits in actual optogenetic experiments. We used thermal imaging to directly measure such temperature rises at the surface of live mouse brains during laser illumination with wavelengths and intensities typical for optogenetics. We then modeled the temperature rise with a simple logarithmic model. Our results indicate that previous finite-element models can underestimate temperature increases by an order of magnitude. We validate our empirical model by predicting the temperature rise caused by pulsed stimulation paradigms. These predictions fit closely to the empirical data and constitute a better estimate of real temperature increases. Additionally, we provide a web-based app for easy calculation that can be used as a tool for safe design of optogenetic experiments.

Functional near-infrared spectroscopy (fNIRS) was used to examine hemodynamic responses in the prefrontal cortex (PFC) during noxious thermal pain, induced by thermal stimulations over three different body sites over the right forearm, right temporomandibular joint, and left forearm. Functional NIRS measurements were obtained from three groups of healthy volunteers, one group for each body region. Each group was subjected to both low-pain stimulation (LPS) and high-pain stimulation (HPS) by a 16  ×  16  mm² thermode of a temperature-controlled thermal stimulator over the respective three body sites. Our results showed that HPS given at three sites induced significant increases (p  <  0.05) in oxy-hemoglobin concentration (ΔHbO) in the PFC with similar temporal patterns in relatively spread PFC areas. In contrast, LPS did not cause any significant ΔHbO in the PFC of any subject group. Our observed PFC activations induced by acute HPS were generally consistent with previous reports by fMRI studies. The study also found a peculiar global trend of postpain deactivation in the PFC, which is attributed to global vasoconstriction due to acute nocuous pain. Overall, these results indicate that hemodynamic activities in PFC exhibit consistent temporal and spatial patterns in response to acute thermal stimulation given across all three body sites.

Diffuse optical tomography (DOT) has recently proved useful for detecting whole-brain oxygenation changes in preterm and term newborns’ brains. The data recording phase in prior explorations was limited up to a maximum of a couple of hours, a time dictated by the need to minimize skin damage caused by the protracted contact with optode holders and interference with concomitant clinical/nursing procedures. In an attempt to extend the data recording phase, we developed a new custom-made cap for multimodal DOT and electroencephalography acquisitions for the neonatal population. The cap was tested on a preterm neonate (28 weeks gestation) for a 7-day continuous monitoring period. The cap was well tolerated by the neonate, who did not suffer any evident discomfort and/or skin damage. Montage and data acquisition using our cap was operated by an attending nurse with no difficulty. DOT data quality was remarkable, with an average of 92% of reliable channels, characterized by the clear presence of the heartbeat in most of them.