Significance: Over the past decade, laser-based digital holographic microscopy (DHM), an important approach in the field of quantitative-phase imaging techniques, has become a significant label-free modality for live-cell imaging and used particularly in cellular neuroscience. However, coherent noise remains a major drawback for DHM, significantly limiting the possibility to visualize neuronal processes and precluding important studies on neuronal connectivity.

Aim: The goal is to develop a DHM technique able to sharply visualize thin neuronal processes.

Approach: By combining a wavelength-tunable light source with the advantages of hologram numerical reconstruction of DHM, an approach called polychromatic DHM (P-DHM), providing OPD images with drastically decreased coherent noise, was developed.

Results: When applied to cultured neuronal networks with an air microscope objective (20  ×  , 0.8 NA), P-DHM shows a coherent noise level typically corresponding to 1 nm at the single-pixel scale, in agreement with the 1  /  N-law, allowing to readily visualize the 1-μm-wide thin neuronal processes with a signal-to-noise ratio of ∼5.

Conclusions: Therefore, P-DHM represents a very promising label-free technique to study neuronal connectivity and its development, including neurite outgrowth, elongation, and branching.

Significance: Cellular layering is a hallmark of the mammalian neocortex with layer and cell type-specific connections within the cortical mantle and subcortical connections. A key challenge in studying circuit function within the neocortex is to understand the spatial and temporal patterns of information flow between different columns and layers.

Aim: We aimed to investigate the three-dimensional (3D) layer- and area-specific interactions in mouse cortex in vivo.

Approach: We applied a new promising neuroimaging method—fluorescence laminar optical tomography in combination with voltage-sensitive dye imaging (VSDi). VSDi is a powerful technique for interrogating membrane potential dynamics in assemblies of cortical neurons, but it is traditionally used for two-dimensional (2D) imaging. Our mesoscopic technique allows visualization of neuronal activity in a 3D manner with high temporal resolution.

Results: We first demonstrated the depth-resolved capability of 3D mesoscopic imaging technology in Thy1-ChR2-YFP transgenic mice. Next, we recorded the long-range functional projections between sensory cortex (S1) and motor cortex (M1) in mice, in vivo, following single whisker deflection.

Conclusions: The results show that mesoscopic imaging technique has the potential to investigate the layer-specific neural connectivity in the mouse cortex in vivo. Combination of mesoscopic imaging technique with optogenetic control strategy is a promising platform for determining depth-resolved interactions between cortical circuit elements.

Significance: Our study is the first comparison of brain activation patterns during motor tasks across unilateral cerebral palsy (UCP), bilateral cerebral palsy (BCP), and typical development (TD) to elucidate neural mechanisms and inform rehabilitation strategies.

Aim: Cortical activation patterns were compared for distal upper and lower extremity tasks in UCP, BCP, and TD using functional near-infrared spectroscopy (fNIRS) and related to functional severity.

Approach: Individuals with UCP (n  =  10, 18.8  ±  6.8  years), BCP (n  =  14, 17.5  ±  9.6  years), and TD (n  =  16, 17.3  ±  9.1  years) participated in this cross-sectional cohort study. The fNIRS was used to noninvasively monitor the hemodynamic response to task-related cortical activation. The block design involved repetitive nondominant hand squeezing and ankle dorsiflexion.

Results: Individuals with UCP demonstrated the highest levels of activation for the squeeze task (UCP  >  BCP q  =  0.049; BCP  >  TD q  <  0.001; and UCP  >  TD q  =  0.001) and more activity in the ipsilateral versus contralateral hemisphere. Individuals with BCP showed the highest levels of cortical activation in the dorsiflexion task (BCP  >  UCP q  <  0.001; BCP  >  TD).

Conclusions: Grouping by CP subtype and manual function or mobility level demonstrated significant differences from TD, even for individuals with the mildest forms of CP. Hemispheric activation patterns showed hypothesized but nonsignificant trends.

Significance: Near-infrared spectroscopy (NIRS) combined with diffuse correlation spectroscopy (DCS) provides a noninvasive approach for monitoring cerebral blood flow (CBF), oxygenation, and oxygen metabolism. However, these methods are vulnerable to signal contamination from the scalp. Our work evaluated methods of reducing the impact of this contamination using time-resolved (TR) NIRS and multidistance (MD) DCS.

Aim: The magnitude of scalp contamination was evaluated by measuring the flow, oxygenation, and metabolic responses to a global hemodynamic challenge. Contamination was assessed by collecting data with and without impeding scalp blood flow.

Approach: Experiments involved healthy participants. A pneumatic tourniquet was used to cause scalp ischemia, as confirmed by contrast-enhanced NIRS, and a computerized gas system to generate a hypercapnic challenge.

Results: Comparing responses acquired with and without the tourniquet demonstrated that the TR-NIRS technique could reduce scalp contributions in hemodynamic signals up to 4 times (r_SD  =  3  cm) and 6 times (r_SD  =  4  cm). Similarly, blood flow responses from the scalp and brain could be separated by analyzing MD DCS data with a multilayer model. Using these techniques, there was no change in metabolism during hypercapnia, as expected, despite large increases in CBF and oxygenation.

Conclusion: NIRS/DCS can accurately monitor CBF and metabolism with the appropriate enhancement to depth sensitivity, highlighting the potential of these techniques for neuromonitoring.

Significance: Systematic studies of the physiological outputs induced by infrared (IR)-mediated inhibition of motor nerves can provide guidance for therapeutic applications and offer critical insights into IR light modulation of complex neural networks.

Aim: We explore the IR-mediated inhibition of action potentials (APs) that either propagate along single axons or are initiated locally and their downstream synaptic transmission responses.

Approach: APs were evoked locally by two-electrode current clamp or at a distance for propagating APs. The neuromuscular transmission was recorded with intracellular electrodes in muscle cells or macro-patch pipettes on terminal bouton clusters.

Results: IR light pulses completely and reversibly terminate the locally initiated APs firing at low frequencies, which leads to blocking of the synaptic transmission. However, IR light pulses only suppress but do not block the amplitude and duration of propagating APs nor locally initiated APs firing at high frequencies. Such suppressed APs do not influence the postsynaptic responses at a distance. While the suppression of AP amplitude and duration is similar for propagating and locally evoked APs, only the former exhibits a 7% to 21% increase in the maximum time derivative of the AP rising phase.

Conclusions: The suppressed APs of motor axons can resume their waveforms after passing the localized IR light illumination site, leaving the muscular and synaptic responses unchanged. IR-mediated modulation on propagating and locally evoked APs should be considered as two separate models for axonal and somatic modulations.

Significance: Dystonia is a dynamic and complex disorder. Real-time analysis of brain activity during motor tasks may increase our knowledge on its pathophysiology. Functional near-infrared spectroscopy (fNIRS) is a noninvasive method that enables the measurement of cortical hemodynamic activity in unconstrained environments.

Aim: We aimed to explore the feasibility of using fNIRS for the study of task-related brain activity in dystonia. Task-related functional magnetic resonance imaging (fMRI) and resting-state functional connectivity were also analyzed.

Approach: Patients with idiopathic right-upper limb dystonia and controls were assessed through nonsimultaneous fMRI and fNIRS during a finger-tapping task. Seed-based connectivity analysis of resting-state fMRI was performed in both groups.

Results: The fMRI results suggest nonspecific activation of the cerebellum and occipital lobe in dystonia patients during the finger-tapping task with the affected hand. Moreover, fNIRS data show lower activation in terms of oxyhemoglobin and total hemoglobin in the frontal, ipsilateral cortex, and somatosensory areas during this task. In dystonia, both fMRI and fNIRS data resulted in hypoactivation of the frontal cortex during finger tapping with both hands simultaneously. Resting-state functional connectivity analysis suggests that the cerebellar somatomotor network in dystonia has an increased correlation with the medial prefrontal cortex and the paracingulate gyrus.

Conclusions: These data suggest that unbalanced activation of the cerebellum, somatosensory, and frontal cortical areas are associated with dystonia. To our knowledge, this is the first study using fNIRS to explore the pathophysiology of dystonia. We show that fNIRS and fMRI are complementary methods and highlight the potential of fNIRS for the study of dystonia and other movement disorders as it can overcome movement restrictions, enabling experiments in more naturalistic conditions.

Significance: The optical properties of biological samples provide information about the structural characteristics of the tissue and any changes arising from pathological conditions. Optical coherence tomography (OCT) has proven to be capable of extracting tissue’s optical properties using a model that combines the exponential decay due to tissue scattering and the axial point spread function that arises from the confocal nature of the detection system, particularly for higher numerical aperture (NA) measurements. A weakness in estimating the optical properties is the inter-parameter cross-talk between tissue scattering and the confocal parameters defined by the Rayleigh range and the focus depth.

Aim: In this study, we develop a systematic method to improve the characterization of optical properties with high-NA OCT.

Approach: We developed a method that spatially parameterizes the confocal parameters in a previously established model for estimating the optical properties from the depth profiles of high-NA OCT.

Results: The proposed parametrization model was first evaluated on a set of intralipid phantoms and then validated using a low-NA objective in which cross-talk from the confocal parameters is negligible. We then utilize our spatially parameterized model to characterize optical property changes introduced by a tissue index matching process using a simple immersion agent, 2,2’-thiodiethonal.

Conclusions: Our approach improves the confidence of parameter estimation by reducing the degrees of freedom in the non-linear fitting model.

Significance: Cerebral oxygenation changes in the superior, middle, and medial gyri were used to elucidate spatial impairments of autonomic hemodynamic recovery during the head-up tilt table test (HUTT) in Parkinson’s disease (PD) patients with orthostatic intolerance (OI) symptoms.

Aim: To analyze dynamic oxygenation changes during the HUTT and classify PD patients with OI symptoms using clinical and oxygenation features.

Approach: Thirty-nine PD patients with OI symptoms [10: orthostatic hypotension (PD-OH); 29: normal HUTT results (PD-NOR)] and seven healthy controls (HCs) were recruited. Prefrontal oxyhemoglobin (HbO) changes during the HUTT were reconstructed with diffuse optical tomography and segmented using the automated anatomical labeling system. Decision trees were used for classification.

Results: HCs and PD-NOR patients with positive rates of HbO change (PD-POS) showed the greatest HbO recovery in the superior frontal gyrus (SFG) during tilt. PD-OH and PD-NOR patients with negative rates of HbO change (PD-NEG) showed asymmetric reoxygenation. The classification accuracy was 89.4% for PD-POS versus PD-NEG, 71% for PD-NOR versus PD-OH, and 55.8% for PD-POS versus PD-NEG versus PD-OH. The oxygenation features were more discriminative than the clinical features.

Conclusions: PD-OH showed decreased right SFG function, which may be associated with impaired compensatory autonomic responses to orthostatic stress.

Significance: Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation.

Aim: We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively.

Approach: A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity.

Results: Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging.

Conclusions: A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.

Significance: Speech processing tasks can be used to assess the integrity and health of many functional and structural aspects of the brain. Despite the potential merits of such behavioral tests as clinical assessment tools, however, the underlying neural substrates remain relatively unclear.

Aim: We aimed to obtain a more in-depth portrait of hemispheric asymmetry during dichotic listening tasks at the level of the prefrontal cortex, where prior studies have reported inconsistent results.

Approach: To avoid central confounds that limited previous studies, we used diffuse correlation spectroscopy to optically monitor cerebral blood flow (CBF) in the dorsolateral prefrontal cortex during dichotic listening tasks in human subjects.

Results: We found that dichotic listening tasks elicited hemispheric asymmetries in both amplitude as well as kinetics. When listening task blocks were repeated, there was an accommodative reduction in the response amplitude of the left, but not the right hemisphere.

Conclusions: These heretofore unobserved trends depict a more nuanced portrait of the functional asymmetry that has been observed previously. To our knowledge, these results additionally represent the first direct measurements of CBF during a speech processing task recommended by the American Speech-Language-Hearing Association for diagnosing auditory processing disorders.

Significance: Solutions for group-level analysis of connectivity from fNIRS observations exist, but groupwise explorative analysis with classical solutions is often cumbersome. Manifold-based solutions excel at data exploration, but there are infinite surfaces crossing the observations cloud of points.

Aim: We aim to provide a systematic choice of surface for a manifold-based analysis of connectivity at group level with small surface interpolation error.

Approach: This research introduces interpolated functional manifold (IFM). IFM builds a manifold from reconstructed changes in concentrations of oxygenated ΔcHbO₂ and reduced ΔcHbR hemoglobin species by means of radial basis functions (RBF). We evaluate the root mean square error (RMSE) associated to four families of RBF. We validated our model against psychophysiological interactions (PPI) analysis using the Jaccard index (JI). We demonstrate the usability in an experimental dataset of surgical neuroergonomics.

Results: Lowest interpolation RMSE was 1.26e  −  4  ±  1.32e  −  8 for ΔcHbO₂ [A.U.] and 4.30e  −  7  ±  2.50e  −  13 [A.U.] for ΔcHbR. Agreement with classical group analysis was JI  =  0.89  ±  0.01 for ΔcHbO₂. Agreement with PPI analysis was JI  =  0.83  ±  0.07 for ΔcHbO₂ and JI  =  0.77  ±  0.06 for ΔcHbR. IFM successfully decoded group differences [ANOVA: ΔcHbO₂: F  (  2,117  )    =  3.07; p  <  0.05; ΔcHbR: F  (  2,117  )    =  3.35; p  <  0.05].

Conclusions: IFM provides a pragmatic solution to the problem of choosing the manifold associated to a cloud of points, facilitating the use of manifold-based solutions for the group analysis of fNIRS datasets.

Significance: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. With a worldwide incidence rate of 2 to 3 per 100,000 people, it accounts for more than 60% of all brain cancers; currently, its 5-year survival rate is <5  %  . GBM treatment relies mainly on surgical resection. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumor detection and guiding the removal of diseased tissues.

Aim: Discriminating healthy brain from GBM tissues in an animal model through the combination of Raman and reflectance spectroscopies.

Approach: EGFP-GL261 cells were injected into the brains of eight laboratory mice for inducing murine GBM in these animals. A multimodal optical fiber probe combining fluorescence, Raman, and reflectance spectroscopy was used to localize in vivo healthy and tumor brain areas and to collect their spectral information.

Results: Tumor areas were localized through the detection of EGFP fluorescence emission. Then, Raman and reflectance spectra were collected from healthy and tumor tissues, and later analyzed through principal component analysis and linear discriminant analysis in order to develop a classification algorithm. Raman and reflectance spectra resulted in 92% and 93% classification accuracy, respectively. Combining together these techniques allowed improving the discrimination between healthy and tumor tissues up to 97%.

Conclusions: These preliminary results demonstrate the potential of multimodal fiber-probe spectroscopy for in vivo label-free detection and delineation of brain tumors, and thus represent an additional, encouraging step toward clinical translation and deployment of fiber-probe spectroscopy.

Significance: Transcranial photobiomodulation (PBM) is a noninvasive neuromodulation technique capable of producing changes in the mitochondrial cytochrome c-oxidase (CCO) activity of neurons. Although the application of PBM in clinical practice and as a neurophysiological tool is increasing, less is known about how different treatment time intervals may result in different outcomes.

Aim: We evaluated the effects of different PBM treatment intervals on brain metabolic activity through the CCO and proto-oncogene expression (c-Fos).

Approach: We studied PBM effects on brain CCO and c-Fos expression in three groups of animals: Control (CN, n  =  8), long interval PBM treatment (LI, n  =  5), and short interval PBM treatment (SI, n  =  5).

Results: Increased CCO activity in the LI group, compared to the SI and CN groups, was found in the prefrontal cortices, dorsal and ventral striatum, and hippocampus. Regarding c-Fos expression, we found a significant increase in the SI group compared to LI and CN, whereas LI showed increased c-Fos expression compared to CN in the cingulate and infralimbic cortices.

Conclusions: We show the effectiveness of different PBM interval schedules in increasing brain metabolic activity or proto-oncogene expression.