The editorial shares a collective vision for Kyiv becoming a regional resource for teaching, practicing, and advancing neurophotonics - with a recipe for developing a summer school program as a forward-looking way to start.

Significance: The cerebrovasculature has become increasingly recognized as a major player in overall brain health and many brain disorders. Although there have been several landmark studies to understand details of these crucially important structures in an anatomically defined area, brain-wide examination of the whole cerebrovasculature, including microvessels, has been challenging. However, emerging techniques, including tissue processing and three-dimensional (3D) microscopy imaging, enable neuroscientists to examine the total vasculature in the entire mouse brain.

Aim: Here, we aim to highlight advances in these high-resolution 3D mapping methods including block-face imaging and light sheet fluorescent microscopy.

Approach: We summarize latest mapping tools to understand detailed anatomical arrangement of the cerebrovascular network and the organizing principles of the neurovascular unit (NVU) as a whole.

Results: We discuss biological insights gained from studies using these imaging methods and how these tools can be used to advance our understanding of the cerebrovascular network and related cell types in the entire brain.

Conclusions: This review article will help to understand recent advance in high-resolution NVU mapping in mice and provide perspective on future studies.

Extracellular vesicles (EVs) are nanoparticles (30 to 1000 nm in diameter) surrounded by a lipid-bilayer which carry bioactive molecules between local and distal cells and participate in intercellular communication. Because of their small size and heterogenous nature they are challenging to characterize. Here, we discuss commonly used techniques that have been employed to yield information about EV size, concentration, mechanical properties, and protein content. These include dynamic light scattering, nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, atomic force microscopy, western blotting, and optical methods including super-resolution microscopy. We also introduce an innovative technique for EV characterization which involves immobilizing EVs on a microscope slide before staining them with antibodies targeting EV proteins, then using the reflectance mode on a confocal microscope to locate the EV plane. By then switching to the microscope’s fluorescence mode, immunostained EVs bearing specific proteins can be identified and the heterogeneity of an EV preparation can be determined. This approach does not require specialist equipment beyond the confocal microscopes that are available in many cell biology laboratories, and because of this, it could become a complementary approach alongside the aforementioned techniques to identify molecular heterogeneity in an EV preparation before subsequent analysis requiring specialist apparatus.

Significance: Whether or not capillary pericytes contribute to blood flow regulation in the brain and retina has long been debated. This was partly caused by failure of detecting the contractile protein α-smooth muscle actin (α-SMA) in capillary pericytes.

Aim: The aim of this review is to summarize recent developments in detecting α-SMA and contractility in capillary pericytes and the relevant literature on the biology of actin filaments.

Results: Evidence suggests that for visualization of the small amounts of α-SMA in downstream mid-capillary pericytes, actin depolymerization must be prevented during tissue processing. Actin filaments turnover is mainly based on de/re-polymerization rather than transcription of the monomeric form, hence, small amounts of α-SMA mRNA may evade detection by transcriptomic studies. Similarly, transgenic mice expressing fluorescent reporters under the α-SMA promoter may yield low fluorescence due to limited transcriptional activity in mid-capillary pericytes. Recent studies show that pericytes including mid-capillary ones express several actin isoforms and myosin heavy chain type 11, the partner of α-SMA in mediating contraction. Emerging evidence also suggests that actin polymerization in pericytes may have a role in regulating the tone of downstream capillaries.

Conclusions: With guidance of actin biology, innovative labeling and imaging techniques can reveal the molecular machinery of contraction in pericytes.

Significance: Functional brain imaging in awake animal models is a popular and powerful technique that allows the investigation of neurovascular coupling (NVC) under physiological conditions. However, ubiquitous facial and body motions (fidgeting) are prime drivers of spontaneous fluctuations in neural and hemodynamic signals. During periods without movement, animals can rapidly transition into sleep, and the hemodynamic signals tied to arousal state changes can be several times larger than sensory-evoked responses. Given the outsized influence of facial and body motions and arousal signals in neural and hemodynamic signals, it is imperative to detect and monitor these events in experiments with un-anesthetized animals.

Aim: To cover the importance of monitoring behavioral state in imaging experiments using un-anesthetized rodents, and describe how to incorporate detailed behavioral and physiological measurements in imaging experiments.

Approach: We review the effects of movements and sleep-related signals (heart rate, respiration rate, electromyography, intracranial pressure, whisking, and other body movements) on brain hemodynamics and electrophysiological signals, with a focus on head-fixed experimental setup. We summarize the measurement methods currently used in animal models for detection of those behaviors and arousal changes. We then provide a guide on how to incorporate this measurements with functional brain imaging and electrophysiology measurements.

Results: We provide a how-to guide on monitoring and interpreting a variety of physiological signals and their applications to NVC experiments in awake behaving mice.

Conclusion: This guide facilitates the application of neuroimaging in awake animal models and provides neuroscientists with a standard approach for monitoring behavior and other associated physiological parameters in head-fixed animals.

Significance: A long-standing challenge of the blood vessel tail artifacts along the axial direction prevents optical coherence tomography angiography (OCTA) for a comprehensive three-dimensional (3D) vascular mapping. Addressing the blood vessel tail artifacts issue will make OCTA to be a real 3D blood vessel structural imaging technique, which in combination with OCT-based blood flow velocity measurements will pave the way for a simpler and robust 3D imaging of the capillary transit time, one important parameter for the evaluation of micro circulation.

Approach: We first described the basic principles of OCTA imaging, discussed the origin of blood vessel tail artifacts in an OCTA image, then reviewed the existing OCTA techniques for tail artifacts suppression, and at last we envisioned the potential solutions for effective OCTA tail artifacts suppression.

Results: The origin of blood vessel tail artifacts is due to the multiple scattering of photons with flowing red blood cells, which elongates the light path of the dynamic signal from vessel lumen to the tail regions. High numerical aperture implementation, subtraction-based post-data processing, Hessian filtering, and high acquisition rate-based dynamic analysis methods have been proposed to address the blood vessel tail artifacts issue in OCTA.

Conclusions: High acquisition rate-based dynamic analysis in combination with Hessian filtering have the potential to effectively suppress the blood vessel tail artifacts and in the meantime preserve flows in small vessels within the tail region, providing real 3D OCTA imaging of blood vessel structures.

Astrocytes integrate information from neurons and the microvasculature to coordinate brain activity and metabolism. Using a variety of calcium-dependent cellular mechanisms, these cells impact numerous aspects of neurophysiology in health and disease. Astrocyte calcium signaling is highly diverse, with complex spatiotemporal features. Here, we review astrocyte calcium dynamics and the optical imaging tools used to measure and analyze these events. We briefly cover historical calcium measurements, followed by our current understanding of how calcium transients relate to the structure of astrocytes. We then explore newer photonics tools including super-resolution techniques and genetically encoded calcium indicators targeted to specific cellular compartments and how these have been applied to astrocyte biology. Finally, we provide a brief overview of analysis software used to accurately quantify the data and ultimately aid in our interpretation of the various functions of astrocyte calcium transients.

Significance: Laser speckle contrast imaging (LSCI) has emerged as a promising tool for intraoperative cerebral blood flow (CBF) monitoring because it produces real-time full-field blood flow maps noninvasively and label free.

Aim: We aim to demonstrate the ability of LSCI to continuously visualize blood flow during neurovascular procedures.

Approach: LSCI hardware was attached to the surgical microscope and did not interfere with the normal operation of the microscope. To more easily visualize CBF in real time, LSCI images were registered with the built-in microscope white light camera such that LSCI images were overlaid on the white light images and displayed to the neurosurgeon continuously in real time.

Results: LSCI was performed throughout each surgery when the microscope was positioned over the patient, providing the surgeon with real-time visualization of blood flow changes before, during, and after aneurysm clipping or arteriovenous malformation (AVM) resection in humans. LSCI was also compared with indocyanine green angiography (ICGA) to assess CBF during aneurysm clipping and AVM surgery; integration of the LSCI hardware with the microscope enabled simultaneous acquisition of LSCI and ICGA.

Conclusions: The results suggest that LSCI can provide continuous and real-time CBF visualization without affecting the surgeon workflow or requiring a contrast agent. The results also demonstrate that LSCI and ICGA provide different, yet complementary information about vessel perfusion.

Significance: Insights into the cellular activity of each member of the neurovascular unit (NVU) is critical for understanding their contributions to neurovascular coupling (NVC)—one of the key control mechanisms in cerebral blood flow regulation. Advances in imaging and genetic tools have enhanced our ability to observe, manipulate and understand the cellular activity of NVU components, namely neurons, astrocytes, microglia, endothelial cells, vascular smooth muscle cells, and pericytes. However, there are still many unresolved questions. Since astrocytes are considered electrically unexcitable, Ca^2  + signaling is the main parameter used to monitor their activity. It is therefore imperative to study astrocytic Ca^2  + dynamics simultaneously with vascular activity using tools appropriate for the question of interest.

Aim: To highlight currently available genetic and imaging tools for studying the NVU—and thus NVC—with a focus on astrocyte Ca^2  + dynamics and vascular activity, and discuss the utility, technical advantages, and limitations of these tools for elucidating NVC mechanisms.

Approach: We draw attention to some outstanding questions regarding the mechanistic basis of NVC and emphasize the role of astrocytic Ca^2  + elevations in functional hyperemia. We further discuss commonly used genetic, and optical imaging tools, as well as some newly developed imaging modalities for studying NVC at the cellular level, highlighting their advantages and limitations.

Results: We provide an overview of the current state of NVC research, focusing on the role of astrocytic Ca^2  + elevations in functional hyperemia; summarize recent advances in genetically engineered Ca^2  + indicators, fluorescence microscopy techniques for studying NVC; and discuss the unmet challenges for future imaging development.

Conclusions: Advances in imaging techniques together with improvements in genetic tools have significantly contributed to our understanding of NVC. Many pieces of the puzzle have been revealed, but many more remain to be discovered. Ultimately, optimizing NVC research will require a concerted effort to improve imaging techniques, available genetic tools, and analytical software.

Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothrombosis model, the photosensitizer rose bengal (RB) is systemically introduced into the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of RB is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific, especially in the depth dimension.We developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation.

Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of RB to confine photothrombosis within a vessel of a target depth.

Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), RB is injected into the blood stream. The scanning wavelength is changed to 720 nm, and photothrombosis is induced by scanning the target vessel.

Results: Two-photon depth-targeted single-vessel photothrombosis was achieved with a success rate of 84.9% ± 1.7% and an irradiation duration of <80 s. Attempts without RB (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm.

Conclusions: We described a protocol that achieves depth-targeted single-vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.

Significance: Fibroblasts are found associated with blood vessels in various locations across the central nervous system (CNS): in the meninges, the choroid plexus, and in the parenchyma within perivascular spaces. CNS fibroblasts have been characterized using transcriptional profiling and a Col1a1-GFP mouse line used to identify CNS fibroblasts in vivo; however, we still know very little regarding their functions and identity.

Aim: Current methods for visualizing CNS fibroblasts are lacking and, in particular, prevent adequate assessment of fibroblast–vessel interactions. We aimed to develop new ways to visualize CNS fibroblasts in greater detail.

Approach: Here, we describe methods for whole mount visualization of meningeal and choroid plexus fibroblasts, and CUBIC optical tissue clearing methods for visualization of parenchymal vessel-associated fibroblasts.

Results: We show that these methods can be used for visualization of vessel-fibroblast interactions in these CNS structures and provide significant improvement over traditional sectioning and staining methods. In addition, we can combine these techniques with immunohistochemistry methods for labeling different cell types in the meninges and blood vasculature as well as EdU-based cell proliferation assays.

Conclusions: We expect these methods will advance studies of CNS fibroblast development and functions in homeostasis, injury, and disease.

Significance: There is a longstanding recommendation within the field of fNIRS to use oxygenated (HbO₂) and deoxygenated (HHb) hemoglobin when analyzing and interpreting results. Despite this, many fNIRS studies do focus on HbO₂ only. Previous work has shown that HbO₂ on its own is susceptible to systemic interference and results may mostly reflect that rather than functional activation. Studies using both HbO₂ and HHb to draw their conclusions do so with varying methods and can lead to discrepancies between studies. The combination of HbO₂ and HHb has been recommended as a method to utilize both signals in analysis.

Aim: We present the development of the hemodynamic phase correlation (HPC) signal to combine HbO₂ and HHb as recommended to utilize both signals in the analysis. We use synthetic and experimental data to evaluate how the HPC and current signals used for fNIRS analysis compare.

Approach: About 18 synthetic datasets were formed using resting-state fNIRS data acquired from 16 channels over the frontal lobe. To simulate fNIRS data for a block-design task, we superimposed a synthetic task-related hemodynamic response to the resting state data. This data was used to develop an HPC-general linear model (GLM) framework. Experiments were conducted to investigate the performance of each signal at different SNR and to investigate the effect of false positives on the data. Performance was based on each signal’s mean T-value across channels. Experimental data recorded from 128 participants across 134 channels during a finger-tapping task were used to investigate the performance of multiple signals [HbO₂, HHb, HbT, HbD, correlation-based signal improvement (CBSI), and HPC] on real data. Signal performance was evaluated on its ability to localize activation to a specific region of interest.

Results: Results from varying the SNR show that the HPC signal has the highest performance for high SNRs. The CBSI performed the best for medium-low SNR. The next analysis evaluated how false positives affect the signals. The analyses evaluating the effect of false positives showed that the HPC and CBSI signals reflect the effect of false positives on HbO₂ and HHb. The analysis of real experimental data revealed that the HPC and HHb signals provide localization to the primary motor cortex with the highest accuracy.

Conclusions: We developed a new hemodynamic signal (HPC) with the potential to overcome the current limitations of using HbO₂ and HHb separately. Our results suggest that the HPC signal provides comparable accuracy to HHb to localize functional activation while at the same time being more robust against false positives.

Significance: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development.

Aim: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin’s head and tail. FRET efficiency decreases with increased tension across vinculin.

Approach: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones.

Results: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod (28.5  ±  3.6  %  ) in growth cones was higher than the mean FRET efficiency of VinTS (24.6  ±  2  %  ) and VinTL (25.8  ±  1.8  %  ) (p  <  ^{10  −  6}). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant (p  <  ^{10  −  3}), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod.

Conclusions: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.

Significance: Functional near-infrared spectroscopy (fNIRS) is a noninvasive technique for measuring hemodynamic changes in the human cortex related to neural function. Due to its potential for miniaturization and relatively low cost, fNIRS has been proposed for applications, such as brain–computer interfaces (BCIs). The relatively large magnitude of the signals produced by the extracerebral physiology compared with the ones produced by evoked neural activity makes real-time fNIRS signal interpretation challenging. Regression techniques incorporating physiologically relevant auxiliary signals such as short separation channels are typically used to separate the cerebral hemodynamic response from the confounding components in the signal. However, the coupling of the extra-cerebral signals is often noninstantaneous, and it is necessary to find the proper delay to optimize nuisance removal.

Aim: We propose an implementation of the Kalman filter with time-embedded canonical correlation analysis for the real-time regression of fNIRS signals with multivariate nuisance regressors that take multiple delays into consideration.

Approach: We tested our proposed method on a previously acquired finger tapping dataset with the purpose of classifying the neural responses as left or right.

Results: We demonstrate computationally efficient real-time processing of 24-channel fNIRS data (400 samples per second per channel) with a two order of selective magnitude decrease in cardiac signal power and up to sixfold increase in the contrast-to-noise ratio compared with the nonregressed signals.

Conclusion: The method provides a way to obtain better distinction of brain from non-brain signals in real time for BCI application with fNIRS.

Significance: Functional near-infrared spectroscopy (fNIRS) enables measuring the brain activity of two subjects while they interact, i.e., the hyperscanning approach.

Aim: In our exploratory study, we extended classical fNIRS hyperscanning by adding systemic physiological measures to obtain systemic physiology augmented fNIRS (SPA-fNIRS) hyperscanning while blocking and not blocking the visual communication between the subjects. This approach enables access brain-to-brain, brain-to-body, and body-to-body coupling between the subjects simultaneously.

Approach: Twenty-four pairs of subjects participated in the experiment. The paradigm consisted of two subjects that sat in front of each other and had their eyes closed for 10 min, followed by a phase of 10 min where they made eye contact. Brain and body activity was measured continuously by SPA-fNIRS.

Results: Our study shows that making eye contact for a prolonged time causes significant changes in brain-to-brain, brain-to-body, and body-to-body coupling, indicating that eye contact is followed by entrainment of the physiology between subjects. Subjects that knew each other generally showed a larger trend to change between the two conditions.

Conclusions: The main point of this study is to introduce a new framework to investigate brain-to-brain, body-to-body, and brain-to-body coupling through a simple social experimental paradigm. The study revealed that eye contact leads to significant synchronization of spontaneous activity of the brain and body physiology. Our study is the first that employed the SPA-fNIRS approach and showed its usefulness to investigate complex interpersonal physiological changes.