Calcium imaging is emerging as a popular technique in neuroscience. A major reason is that intracellular calcium transients are reflections of electrical events in neurons. For example, calcium influx in the soma and axonal boutons accompanies spiking activity, whereas elevations in dendrites and dendritic spines are associated with synaptic inputs and local regenerative events. However, calcium transients have complex spatiotemporal dynamics, and since most optical methods visualize only one of the somatic, axonal, and dendritic compartments, a straightforward inference of the underlying electrical event is typically challenging. We highlight experiments that have directly calibrated in vivo calcium signals recorded using fluorescent indicators against electrophysiological events. We address commonly asked questions such as: Can calcium imaging be used to characterize neurons with high firing rates? Can the fluorescent signal report a decrease in spiking activity? What is the evidence that calcium transients in subcellular compartments correspond to distinct presynaptic axonal and postsynaptic dendritic events? By reviewing the empirical evidence and limitations, we suggest that, despite some caveats, calcium imaging is a versatile method to characterize a variety of neuronal events in vivo.

Monitoring speech tasks with functional near-infrared spectroscopy (fNIRS) enables investigation of speech production mechanisms and informs treatment strategies for speech-related disorders such as stuttering. Unfortunately, due to movement of the temporalis muscle, speech production can induce relative movement between probe optodes and skin. These movements generate motion artifacts during speech tasks. In practice, spurious hemodynamic responses in functional activation signals arise from lack of information about the consequences of speech-related motion artifacts, as well as from lack of standardized processing procedures for fNIRS signals during speech tasks. To this end, we characterize the effects of speech production on fNIRS signals, and we introduce a systematic analysis to ameliorate motion artifacts. The study measured 50 healthy subjects performing jaw movement (JM) tasks and found that JM produces two different patterns of motion artifacts in fNIRS. To remove these unwanted contributions, we validate a hybrid motion-correction algorithm based sequentially on spline interpolation and then wavelet filtering. We compared performance of the hybrid algorithm with standard algorithms based on spline interpolation only and wavelet decomposition only. The hybrid algorithm corrected 94% of the artifacts produced by JM, and it did not lead to spurious responses in the data. We also validated the hybrid algorithm during a reading task performed under two different conditions: reading aloud and reading silently. For both conditions, we observed significant cortical activation in brain regions related to reading. Moreover, when comparing the two conditions, good agreement of spatial and temporal activation patterns was found only when data were analyzed using the hybrid approach. Overall, the study demonstrates a standardized processing scheme for fNIRS data during speech protocols. The scheme decreases spurious responses and intersubject variability due to motion artifacts.

We used a new multimodal imaging system that combines optical coherence microscopy and brightfield microscopy. Using this in vivo brain monitoring approach and cranial window implantation, we three-dimensionally visualized the vascular network during thrombosis, with high temporal (18 s) and spatial (axial, 2.5  μm; lateral, 2.2  μm) resolution. We used a modified mouse model of photochemical thromboembolic stroke in order to more accurately parallel human stroke. Specifically, we applied green laser illumination to focally occlude a branch of the middle cerebral artery. Despite the recanalization of the superficial arteries at 24 h after stroke, no blood flow was detected in the small vessels within deeper regions. Moreover, after 24 h of stroke progression, scattering signal enhancement was observed within the stroke region. We also evaluated the infarct extent and shape histologically. In summary, we present a novel approach for real-time mouse brain monitoring and ischemic variability analysis. This multimodal imaging method permits the analysis of thrombosis progression and reperfusion. Additionally and importantly, the system could be used to study the effect of poststroke drug treatments on blood flow in small arteries and capillaries of the brain.

Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.

Significance: Cortically implanted microelectrode arrays provide a direct interface with neuronal populations and are used to restore movement capabilities and provide sensory feedback to patients with paralysis or amputation. Penetrating electrodes experience high rates of signal degradation within the first year that limit effectiveness and lead to eventual device failure.

Aim: To assess vascular and neuronal changes over time in mice with implanted electrodes and examine the contribution of the brain tissue response to electrode performance.

Approach: We used a multimodal approach combining in vivo electrophysiology and subcellular-level optical imaging.

Results: At acute timescales, we observed structural damage from the mechanical trauma of electrode insertion, evidenced by severed dendrites in the electrode path and local hypofluorescence. Superficial vessel growth and remodeling occurred within the first few weeks in both electrode-implanted and window-only animals, but the deeper capillary growth evident in window-only animals was suppressed in electrode-implanted animals. After longer implantation periods, there was evidence of degeneration of transected dendrites superficial to the electrode path and localized neuronal cell body loss, along with deep vascular velocity changes near the electrode. Total spike rate (SR) across all animals reached a peak between 3 and 9 months postimplantation, then decreased. The local field potential signal remained relatively constant for up to 6 months, particularly in the high-gamma band, indicating long-term electrode viability and neuronal functioning at further distances from the electrode, but it showed a reduction in some animals at later time points. Most importantly, we found that progressive high-gamma and SR reductions both correlate positively with localized cell loss and decreasing capillary density within 100  μm of the electrode.

Conclusions: This multifaceted approach provided a more comprehensive picture of the ongoing biological response at the brain–electrode interface than can be achieved with postmortem histology alone and established a real-time relationship between electrophysiology and tissue damage.

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.

Significance. Recent Alzheimer’s disease (AD) patient studies have focused on retinal analysis, as the retina is the only part of the central nervous system that can be imaged noninvasively by optical methods. However, as this is a relatively new approach, the occurrence and role of retinal pathological features are still debated.

Aim. The retina of an APP/PS1 mouse model was investigated using multicontrast optical coherence tomography (OCT) in order to provide a documentation of what was observed in both transgenic and wild-type mice.

Approach. Both eyes of 24 APP/PS1 transgenic mice (age: 45 to 104 weeks) and 15 age-matched wild-type littermates were imaged by the custom-built OCT system. At the end of the experiment, retinas and brains were harvested from a subset of the mice (14 transgenic, 7 age-matched control) in order to compare the in vivo results to histological analysis and to quantify the cortical amyloid beta plaque load.

Results. The system provided a combination of standard reflectivity data, polarization-sensitive data, and OCT angiograms. Qualitative and quantitative information from the resultant OCT images was extracted on retinal layer thickness and structure, presence of hyper-reflective foci, phase retardation abnormalities, and retinal vasculature.

Conclusions. Although multicontrast OCT revealed abnormal structural properties and phase retardation signals in the retina of this APP/PS1 mouse model, the observations were very similar in transgenic and control mice.

Significance: Natural brain adaptations often involve changes in synaptic strength. The artificial manipulations can help investigate the role of synaptic strength in a specific brain circuit not only in various physiological phenomena like correlated neuronal firing and oscillations but also in behaviors. High- and low-frequency stimulation at presynaptic sites has been used widely to induce long-term potentiation (LTP) and depression. This approach is effective in many brain areas but not in the basolateral amygdala (BLA) because the robust local GABAergic tone inside BLA restricts synaptic plasticity.

Aim: We aimed at identifying the subclass of GABAergic neurons that gate LTP in the BLA afferents from the dorsomedial prefrontal cortex (dmPFC).

Approach: Chemogenetic or optogenetic suppression of specific GABAergic neurons in BLA was combined with high-frequency stimulation of the BLA afferents as a method for LTP induction.

Results: Chemogenetic suppression of somatostatin-positive interneurons (Sst-INs) enabled the ex vivo LTP by high-frequency stimulation of the afferent but the suppression of parvalbumin-positive interneurons (PV-INs) did not. Moreover, optogenetic suppression of Sst-INs with Arch also enabled LTP of the dmPFC-BLA synapses, both ex vivo and in vivo.

Conclusions: These findings reveal that Sst-INs but not PV-INs gate LTP in the dmPFC-BLA pathway and provide a method for artificial synaptic facilitation in BLA.

Significance: Functional near-infrared spectroscopy (fNIRS) has become an important research tool in studying human brains. Accurate quantification of brain activities via fNIRS relies upon solving computational models that simulate the transport of photons through complex anatomy.

Aim: We aim to highlight the importance of accurate anatomical modeling in the context of fNIRS and propose a robust method for creating high-quality brain/full-head tetrahedral mesh models for neuroimaging analysis.

Approach: We have developed a surface-based brain meshing pipeline that can produce significantly better brain mesh models, compared to conventional meshing techniques. It can convert segmented volumetric brain scans into multilayered surfaces and tetrahedral mesh models, with typical processing times of only a few minutes and broad utilities, such as in Monte Carlo or finite-element-based photon simulations for fNIRS studies.

Results: A variety of high-quality brain mesh models have been successfully generated by processing publicly available brain atlases. In addition, we compare three brain anatomical models—the voxel-based brain segmentation, tetrahedral brain mesh, and layered-slab brain model—and demonstrate noticeable discrepancies in brain partial pathlengths when using approximated brain anatomies, ranging between −1.5  %   to 23% with the voxelated brain and 36% to 166% with the layered-slab brain.

Conclusion: The generation and utility of high-quality brain meshes can lead to more accurate brain quantification in fNIRS studies. Our open-source meshing toolboxes “Brain2Mesh” and “Iso2Mesh” are freely available at http://mcx.space/brain2mesh.

Significance: Major depressive disorder (MDD) affects over 40 million U.S. adults in their lifetime. Transcranial photobiomodulation (t-PBM) has been shown to be effective in treating MDD, but the current treatment dosage does not account for head and brain anatomical changes due to aging.

Aim: We study effective t-PBM dosage and its variations across age groups using state-of-the-art Monte Carlo simulations and age-dependent brain atlases ranging between 5 and 85 years of age.

Approach: Age-dependent brain models are derived from 18 MRI brain atlases. Two extracranial source positions, F3–F4 and Fp1–Fpz–Fp2 in the EEG 10–20 system, are simulated at five selected wavelengths and energy depositions at two MDD-relevant cortical regions—dorsolateral prefrontal cortex (dlPFC) and ventromedial prefrontal cortex (vmPFC)—are quantified.

Results: An overall decrease of energy deposition was found with increasing age. A strong negative correlation between the thickness of extracerebral tissues (ECT) and energy deposition was observed, suggesting that increasing ECT thickness over age is primarily responsible for reduced energy delivery. The F3–F4 position appears to be more efficient in reaching dlPFC compared to treating vmPFC via the Fp1–Fpz–Fp2 position.

Conclusions: Quantitative simulations revealed age-dependent light delivery across the lifespan of human brains, suggesting the need for personalized and age-adaptive t-PBM treatment planning.

Significance: The expanding field of human social interaction is enabled by functional near-infrared spectroscopy (fNIRS) that acquires hemodynamic signals during live two-person interactions. These advances call for development of methods to quantify interactive processes.

Aim: Wavelet coherence analysis has been applied to cross-brain neural coupling. However, fNIRS-specific computations have not been explored. This investigation determines the effects of global mean removal, wavelet equation, and choice of oxyhemoglobin versus deoxyhemoglobin signals.

Approach: We compare signals with a known coherence with acquired signals to determine optimal computational approaches. The known coherence was calculated using three visual stimulation sequences of a contrast-reversing checkerboard convolved with the canonical hemodynamic response function. This standard was compared with acquired human fNIRS responses within visual cortex using the same sequences.

Results: Observed coherence was consistent with known coherence with highest correlations within the wavelength range between 10 and 20 s. Removal of the global mean improved the correlation irrespective of the specific equation for wavelet coherence, and the oxyhemoglobin signal was associated with a marginal correlation advantage.

Conclusions: These findings provide both methodological and computational guidance that enhances the validity and interpretability of wavelet coherence analysis for fNIRS signals acquired during live social interactions.

Optogenetics has become an integral tool for studying and dissecting the neural circuitries of the brain using optical control. Recently, it has also begun to be used in the investigation of the spinal cord and peripheral nervous system. However, information on these regions’ optical properties is sparse. Moreover, there is a lack of data on the dependence of light propagation with respect to neural tissue organization and orientation. This information is important for effective simulations and optogenetic planning, particularly in the spinal cord where the myelinated axons are highly organized. To this end, we report experimental measurements for the scattering coefficient, validated with three different methods in both the longitudinal and radial directions of multiple mammalian spinal cords. In our analysis, we find that there is indeed a directional dependence of photon propagation when interacting with organized myelinated axons. Specifically, light propagating perpendicular to myelinated axons in the white matter of the spinal cord produced a measured reduced scattering coefficient (μs′) of 3.52  ±  0.1  mm^−  1, and light that was propagated along the myelinated axons in the white matter produced a measured μs′ of 1.57  ±  0.03  mm^−  1, across the various species considered. This 50% decrease in scattering power along the myelinated axons is observed with three different measurement strategies (integrating spheres, observed transmittance, and punch-through method). Furthermore, this directional dependence in scattering power and overall light attenuation did not occur in the gray matter regions where the myelin organization is nearly random. The acquired information will be integral in preparing future light-transport simulations and in overall optogenetic planning in both the spinal cord and the brain.

Significance: Attention-deficit/hyperactivity disorder (ADHD) is the most common psychological disease in childhood. Currently, widely used neuroimaging techniques require complete body confinement and motionlessness and thus are extremely hard for brain scanning of ADHD children.

Aim: We present resting-state functional near-infrared spectroscopy (fNIRS) as an imaging technique to record spontaneous brain activity in children with ADHD.

Approach: The brain functional connectivity was calculated, and the graph theoretical analysis was further applied to investigate alterations in the global and regional properties of the brain network in the patients. In addition, the relationship between brain network features and core symptoms was examined.

Results: ADHD patients exhibited significant decreases in both functional connectivity and global network efficiency. Meanwhile, the nodal efficiency in children with ADHD was also found to be altered, e.g., increase in the visual and dorsal attention networks and decrease in somatomotor and default mode networks, compared to the healthy controls. More importantly, the disrupted functional connectivity and nodal efficiency significantly correlated with dimensional ADHD scores.

Conclusions: We clearly demonstrate the feasibility and potential of fNIRS-based connectome technique in ADHD or other neurological diseases in the future.

Significance: There are no label-free imaging descriptors related to physiological activity of inner retinal cells in the living human eye. A major reason is that inner retinal neurons are highly transparent and reflect little light, making them extremely difficult to visualize and quantify.

Aim: To measure physiologically-induced optical changes of inner retinal cells despite their challenging optical properties.

Approach: We developed an imaging method based on adaptive optics and optical coherence tomography (AO-OCT) and a suite of postprocessing algorithms, most notably a new temporal correlation method.

Results: We captured the temporal dynamics of entire inner retinal layers, of specific tissue types, and of individual cells across three different timescales from fast (seconds) to extremely slow (one year). Time correlation analysis revealed significant differences in time constant (up to 0.4 s) between the principal layers of the inner retina with the ganglion cell layer (GCL) being the most dynamic. At the cellular level, significant differences were found between individual GCL somas. The mean time constant of the GCL somas (0.69  ±  0.17  s) was   ∼  30  %   smaller than that of nerve fiber bundles and inner plexiform layer synapses and processes. Across longer durations, temporal speckle contrast and time-lapse imaging revealed motion of macrophage-like cells (over minutes) and GCL neuron loss and remodeling (over one year).

Conclusions: Physiological activity of inner retinal cells is now measurable in the living human eye.

Significance: Current approaches to stimulating and recording from the brain have combined electrical or optogenetic stimulation with recording approaches, such as two-photon, electrophysiology (EP), and optical intrinsic signal imaging (OISI). However, we lack a label-free, all-optical approach with high spatial and temporal resolution.

Aim: To develop a label-free, all-optical method that simultaneously manipulates and images brain function using pulsed near-infrared light (INS) and functional optical coherence tomography (fOCT), respectively.

Approach: We built a coregistered INS, fOCT, and OISI system. OISI and EP recordings were employed to validate the fOCT signals.

Results: The fOCT signal was reliable and regional, and the area of fOCT signal corresponded with the INS-activated region. The fOCT signal was in synchrony with the INS onset time with a delay of ∼30  ms. The magnitude of fOCT signal exhibited a linear correlation with the INS radiant exposure. The significant correlation between the fOCT signal and INS was further supported by OISI and EP recordings.

Conclusions: The proposed fiber-based, all-optical INS-fOCT method allows simultaneous stimulation and mapping without the risk of interchannel cross-talk and the requirement of contrast injection and viral transfection and offers a deep penetration depth and high resolution.

Corrected disclosures for the article “Prolonged monitoring of cerebral blood flow and autoregulation with diffuse correlation spectroscopy in neurocritical care patients.”