Time-domain diffuse correlation spectroscopy (TD-DCS) is an emerging noninvasive optical technique with the potential to resolve blood flow (BF) and optical coefficients (reduced scattering and absorption) in depth. Here, we study the effects of finite temporal resolution and gate width in a realistic TD-DCS experiment. We provide a model for retrieving the BF from gated intensity autocorrelations based on the instrument response function, which allows for the use of broad time gates. This, in turn, enables a higher signal-to-noise ratio that is critical for in vivo applications. In numerical simulations, the use of the proposed model reduces the error in the estimated late gate BF from 34% to 3%. Simulations are also performed for a wide set of optical properties and source–detector separations. In a homogeneous phantom experiment, the discrepancy between later gates BF index and ungated BF index is reduced from 37% to 2%. This work not only provides a tool for data analysis but also physical insights, which can be useful for studying and optimizing the system performance.

Modulation of brain state, e.g., by anesthesia, alters the correlation structure of spontaneous activity, especially in the delta band. This effect has largely been attributed to the ∼1  Hz slow oscillation that is characteristic of anesthesia and nonrapid eye movement (NREM) sleep. However, the effect of the slow oscillation on correlation structures and the spectral content of spontaneous activity across brain states (including NREM) has not been comprehensively examined. Further, discrepancies between activity dynamics observed with hemoglobin versus calcium (GCaMP6) imaging have not been reconciled. Lastly, whether the slow oscillation replaces functional connectivity (FC) patterns typical of the alert state, or superimposes on them, remains unclear. Here, we use wide-field calcium imaging to study spontaneous cortical activity in awake, anesthetized, and naturally sleeping mice. We find modest brain state-dependent changes in infraslow correlations but larger changes in GCaMP6 delta correlations. Principal component analysis of GCaMP6 sleep/anesthesia data in the delta band revealed that the slow oscillation is largely confined to the first three components. Removal of these components revealed a correlation structure strikingly similar to that observed during wake. These results indicate that, during NREM sleep/anesthesia, the slow oscillation superimposes onto a canonical FC architecture.

The methods used for digital processing of optical coherence tomography (OCT) and crosspolarization (CP) OCT images are focused on improving the contrast ratio of native structural OCT images. Such advances are particularly important for the intraoperative detection of glioma margins where the visual assessment of OCT images can be difficult and lead to errors. The aim of the study was to investigate the application of optical coefficients obtained from CP OCT data for the differentiation of glial tumorous tissue from a normal brain. Pseudocolor en-face OCT maps based on two optical coefficients (the commonly used rate of attenuation in the cochannel, and in addition, the interchannel attenuation difference) were constructed for normal rat brain coronal cross sections and for brains with a 101.8 rat glioblastoma model. It was shown that the use of optical coefficients significantly increased the available information from the OCT data in comparison with unprocessed images. As a result, this allowed contrasting of the white matter from the gray matter and tumorous tissue ex vivo, and for this purpose, the interchannel attenuation difference worked better. The interchannel attenuation difference values of white matter were at least seven and two times higher than corresponding values of the cortex and tumorous tissue, whereas the same parameter for cochannel attenuation coefficient values of white matter are about 4 and 1.4. However, quantitative analysis shows that both coefficients are suitable for the purpose of glioblastoma detection from normal brain tissue regardless of whether a necrotic component was present (in all compared groups p  <  0.001).

Optical coherence tomography provides volumetric reconstruction of brain structure with micrometer resolution. Gray matter and white matter can be highlighted using conventional and polarization-based contrasts; however, vasculature in ex-vivo fixed brain has not been investigated at large scale due to lack of intrinsic contrast. We present contrast enhancement to visualize the vasculature by perfusing titanium dioxide particles transcardially into the mouse vascular system. The brain, after dissection and fixation, is imaged by a serial optical coherence scanner. Accumulation of particles in blood vessels generates distinguishable optical signals. Among these, the cross-polarization images reveal the vasculature organization remarkably well. The conventional and polarization-based contrasts are still available for probing the gray matter and white matter structures. The segmentation and reconstruction of the vasculature are presented by using a deep learning algorithm. Axonal fiber pathways in the mouse brain are delineated by utilizing the retardance and optic axis orientation contrasts. This is a low-cost method that can be further developed to study neurovascular diseases and brain injury in animal models.

Functional near-infrared spectroscopy (fNIRS) estimates the functional oscillations of oxyhemoglobin and deoxyhemoglobin in the cortex through scalp-located multiwavelength recordings. Hemoglobin oscillations are inferred through temporal changes in continuous-wave (CW) light attenuation. However, because of the diffusive multilayered head tissue structures, the photon path is longer than the source–detector separation, complicating hemoglobin evaluation. This aspect is incorporated in the modified Beer–Lambert law where the source–detector distance is multiplied by the differential pathlength factor (DPF). Since DPF estimation requires photons’ time-of-flight information, DPF is assumed a priori in CW-fNIRS. Importantly, errors in the DPF spectrum induce hemoglobin cross talk, which is detrimental for fNIRS. We propose to estimate subject-specific DPF spectral dependence relying on multidistance high-density measurements. The procedure estimates the effective attenuation coefficient (EAC), which is proportional to the geometric mean of absorption and reduced scattering. Since DPF depends on the scattering-to-absorption ratio, EAC limits the spectral dependence assumption to scattering. This approach was compared to a standard frequency-domain multidistance procedure. A good association between the two methods (r²  =  0.69) was obtained. This approach could estimate low-resolution maps of the DPF spectral dependence through large field of view, high-density systems, reducing hemoglobin cross talk, and increasing fNIRS sensitivity and specificity to brain activity without instrumentation modification.

Sickle cell disease (SCD) is a genetic blood disorder that has profound effects on the brain. Chronic anemia combined with both macro- and microvascular perfusion abnormalities that arise from stenosis or occlusion of blood vessels increased blood viscosity, adherence of red blood cells to the vascular endothelium, and impaired autoregulatory mechanisms in SCD patients all culminate in susceptibility to cerebral infarction. Indeed, the risk of stroke is 250 times higher in children with SCD than in the general population. Unfortunately, while transcranial Doppler ultrasound (TCD) has been widely clinically adopted to longitudinally monitor macrovascular perfusion in these patients, routine clinical screening of microvascular perfusion abnormalities is challenging with current modalities (e.g., positron emission tomography and magnetic resonance imaging) given their high-cost, requirement for sedation in children <6 year, and need for trained personnel. We assess the feasibility of a low-cost, noninvasive optical technique known as diffuse correlation spectroscopy (DCS) to quantify an index of resting-state cortical cerebral blood flow (BFI) in 11 children with SCD along with 11 sex- and age-matched healthy controls. As expected, BFI was significantly higher in SCD subjects compared to healthy controls (p  <  0.001). Within SCD subjects, BFI was inversely proportional to resting-state arterial hemoglobin levels (p  =  0.012), consistent with expected anemia-induced compensatory vasodilation that aims to maintain adequate oxygen delivery to the tissue. Further, in a subset of patients measured with TCD (n  =  7), DCS-measured blood flow was correlated with TCD-measured blood flow velocity in middle cerebral artery (R_s  =  0.68), although the trend was not statistically significant (p  =  0.11). These results are consistent with those of several previous studies using traditional neuroimaging techniques, suggesting that DCS may be a promising low-cost tool for assessment of tissue-level CBF in pediatric SCD.

Measurements of dynamic near-infrared (NIR) light attenuation across the human head together with model-based image reconstruction algorithms allow the recovery of three-dimensional spatial brain activation maps. Previous studies using high-density diffuse optical tomography (HD-DOT) systems have reported improved image quality over sparse arrays. These HD-DOT systems incorporated multidistance overlapping continuous wave measurements that only recover differential intensity attenuation. We investigate the potential improvement in reconstructed image quality due to the additional incorporation of phase shift measurements, which reflect the time-of-flight of the measured NIR light, within the tomographic reconstruction from high-density measurements. To evaluate image reconstruction with and without the additional phase information, we simulated point spread functions across a whole-scalp field of view in 24 subject-specific anatomical models using an experimentally derived noise model. The addition of phase information improves the image quality by reducing localization error by up to 59% and effective resolution by up to 21% as compared to using the intensity attenuation measurements alone. Furthermore, we demonstrate that the phase data enable images to be resolved at deeper brain regions where intensity data fail, which is further supported by utilizing experimental data from a single subject measurement during a retinotopic experiment.

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the “local proteome.” Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

Near-infrared radiation (NIR) has been described as one of the highest-resolution tools for neuromodulation. However, the poor tissue penetration depth of NIR has limited its further application on some of the deeper layer neurons in vivo. A 980-nm short-wavelength NIR (SW-NIR) with high penetration depth was employed, and its inhibitory effect on neurons was investigated in vivo. In experiments, SW-NIR was implemented on the rat’s cochlear nucleus (CN), the auditory pathway was activated by pure-tones through the rat’s external auditory canal, and the neural responses were recorded in the inferior colliculus by a multichannel electrode array. Neural firing rate (FR) and the first spike latency (FSL) were analyzed to evaluate the optically induced neural inhibition. Meanwhile, a two-layered finite element, consisting of a fluid layer and a gray matter layer, was established to model the optically induced temperature changes in CN; different stimulation paradigms were used to compare the inhibitory efficiency of SW-NIR. Results showed that SW-NIR could reversibly inhibit acoustically induced CN neural activities: with the increase of laser radiant exposures energy, neural FR decreased significantly and FSL lengthened steadily. Significant inhibition occurred when the optical pulse stimulated prior to the acoustic stimulus. Results indicated that the inhibition relies on the establishment time of the temperature field. Moreover, our preliminary results suggest that short-wavelength infrared could regulate the activities of neurons beyond the neural tissues laser irradiated through neural networks and conduction in vivo. These findings may provide a method for accurate neuromodulation in vivo.

We present an electrically addressable optrode array capable of delivering light to 181 sites in the brain, each providing sufficient light to optogenetically excite thousands of neurons in vivo, developed with the aim to allow behavioral studies in large mammals. The device is a glass microneedle array directly integrated with a custom fabricated microLED device, which delivers light to 100 needle tips and 81 interstitial surface sites, giving two-level optogenetic excitation of neurons in vivo. Light delivery and thermal properties are evaluated, with the device capable of peak irradiances >80  mW  /  mm² per needle site. The device consists of an array of 181 80  μm  ×  80  μm² microLEDs, fabricated on a 150-μm-thick GaN-on-sapphire wafer, coupled to a glass needle array on a 150-μm thick backplane. A pinhole layer is patterned on the sapphire side of the microLED array to reduce stray light. Future designs are explored through optical and thermal modeling and benchmarked against the current device.

Fiber photometry has been increasingly popular in neuroscience research in freely behaving animals. In combination with genetically encoded calcium indicators, it allows for real-time monitoring of neural activity in neuronal somata, dendrites, and axonal terminals. We developed a multichannel fiber photometry device to map the activity of axonal terminals in a restricted, 100-μm-wide brain region in freely moving mice. This device consists of four bundled multimode fibers, each with a 50-μm core diameter and a scientific complementary metal-oxide semiconductor camera to simultaneously acquire fluorescence. We achieved a sampling rate of 100  frames  /  s and sufficient sensitivity to acquire data from axonal terminals. To avoid interference with neighboring channels, the recording depth of each channel was restricted to <250  μm. Furthermore, the small-core-diameter fibers did not restrict mouse locomotion. Using the Ca^2  + indicator GCaMP5G, we validated the system by recording Ca^2  + signals in axonal terminals from the medial entorhinal cortex layer II to the hippocampal dentate gyrus (DG) in freely moving mice. We detected spatially separated Ca^2  + signals at four different sites in the DG. Therefore, our multichannel fiber photometry device provides a simple but powerful method to functionally map axonal terminals in spatially confined brain areas of freely moving animals.

Large-scale, high-resolution imaging of cerebral hemodynamics is essential for brain research. Uniquely capable of comprehensive quantification of cerebral hemodynamics and oxygen metabolism in rodents based on the endogenous hemoglobin contrast, multiparametric photoacoustic microscopy (PAM) is ideally suited for this purpose. However, the out-of-focus issue due to the uneven surface of the rodent brain results in inaccurate PAM measurements and presents a significant challenge to cortex-wide multiparametric recording. We report a large-scale, high-resolution, multiparametric PAM system based on real-time surface contour extraction and scanning, which avoids the prescan and offline calculation of the contour map required by previously reported contour-scanning strategies. The performance of this system has been demonstrated in both phantoms and the live mouse brain through a thinned-skull window. Side-by-side comparison shows that the real-time contour scanning not only improves the quality of structural images by addressing the out-of-focus issue but also ensures accurate measurements of the concentration of hemoglobin (C_Hb), oxygen saturation of hemoglobin (sO₂), and cerebral blood flow (CBF) over the entire mouse cortex. Furthermore, quantitative analysis reveals how the out-of-focus issue impairs the measurements of C_Hb, sO₂, and CBF.

In a pilot study of 11 healthy adults (24 to 39 years, all male), we characterize the influence of external probe pressure on optical diffuse correlation spectroscopy (DCS) measurements of pulsatile blood flow obtained on the forearm and forehead. For external probe pressure control, a hand inflatable air balloon is inserted between the tissue and an elastic strap. The air balloon is sequentially inflated to achieve a wide range of external probe pressures between 20 and 250 mmHg on the forearm and forehead, which are measured with a flexible pressure sensor underneath the probe. At each probe pressure, the pulsatility index (PI) of arteriole blood flow on the forehead and forearm is measured with DCS (2.1-cm source-detector separation). We observe a strong correlation between probe pressure and PI on the forearm (R  =  0.66, p  <  0.001), but not on the forehead (R  =    −  0.11, p  =  0.4). The forearm measurements demonstrate the sensitivity of the DCS PI to skeletal muscle tissue pressure, whereas the forehead measurements indicate that DCS PI measurements are not sensitive to scalp tissue pressure. Note, in contrast to pulsatility, the time-averaged DCS blood flow index on the forehead was significantly correlated with probe pressure (R  =    −  0.55, p  <  0.001). This pilot data appears to support the initiation of more comprehensive clinical studies on DCS to detect trends in internal pressure in brain and skeletal muscle.

Accurate histopathological diagnosis is essential for facilitating the optimal surgical management of intracranial germinoma. Current intraoperative histological methods are time- and labor-intensive and often produce artifacts. Multiphoton microscopy (MPM) is a label-free imaging technique that can produce intraoperative histological images of fresh, unprocessed surgical specimens. We employ an MPM based on second-harmonic generation and two-photon excited fluorescence microscopy to image fresh, unfixed, and unstained human germinoma specimens. We show that label-free MPM is not only capable of identifying various cells in human germinoma tissue but also capable of revealing the characteristics of germinoma such as granuloma, stromal fibrosis, calcification, as well as the abnormal and uneven structures of blood vessels. In conjunction with custom-developed image-processing algorithms, MPM can further quantify and characterize the extent of stromal fibrosis and calcification. Our results provide insight into how MPM can deliver rapid diagnostic histological data that could inform the surgical management of intracranial germinoma.