Neurophotonics

SignificanceCerebral autoregulation (CA) reflects the dynamic coupling among cerebral blood flow (CBF), intracranial pressure (ICP), and arterial blood pressure (ABP); its failure contributes to secondary brain injury. Existing bedside methods rely on indirect or spatially limited CBF surrogates and cannot resolve microvascular flow dynamics across space, depth, and time. AimTo develop, optimize, and apply a scalable, noncontact time-resolved laser speckle contrast imaging (TR-LSCI) platform for depth-sensitive, high-speed, wide-field CBF imaging during controlled ICP perturbations. ApproachTR-LSCI synchronizes a 20-MHz pulsed laser with a time-gated, single-photon avalanche diode (SPAD) camera (512 x 512 pixels) to detect diffuse photons at varying path lengths, enabling depth-resolved microvascular CBF imaging. Benchtop and mobile TR-LSCI systems were applied in adult rats and a neonatal piglet with synchronized invasive ICP and ABP measurements. ResultsTR-LSCI captured spatially heterogeneous, pulsatile CBF dynamics at up to 52 Hz over large cortical fields of view, with heart rate estimates statistically equivalent to those from ICP and ABP. Multivariable analysis identified reproducible, phase-dependent CA transitions encompassing preserved autoregulation, ABP-driven compensation, and ICP-constrained CBF suppression; notably, CBF alone exhibited distinct phase signatures. ConclusionsTR-LSCI enables dynamic, physiology-informed neurovascular monitoring and supports future bedside CA assessment.

SignificanceLaser speckle contrast imaging (LSCI) is widely used to measure blood flow, but speckle fluctuations may also encode biologically meaningful dynamics beyond perfusion. Foundational studies in dynamic light scattering (DLS) and micro-optical coherence tomography (OCT) have also demonstrated that slow coherent signal fluctuations can arise from energy-dependent intracellular motion in in vitro and ex vivo systems. Building upon these advances, recent work has shown that LSCI has the potential to detect slow speckle dynamics (SSD) correlated with cellular dynamics in vivo. However, the biophysical mechanisms underlying SSD in intact brain tissues remain insufficiently validated. Establishing a mechanistic bridge from controlled ex vivo and in vitro conditions to in vivo brain measurements is critical for translating speckle-based imaging beyond perfusion measurements to enable label-free assessment of cellular and metabolic activity in disease models. AimThe objective of this study is to investigate the biophysical origin of the SSD in vivo and evaluate its sensitivity to intracellular metabolic activity in brain tissue. ApproachWe utilize an epi-illumination LSCI system to measure speckle contrast as a function of camera exposure time and extract characteristic decorrelation time constants. SSD was investigated in acute mouse brain slices, where blood flow is absent, to eliminate vascular confounds. Cellular metabolism was systematically modulated using 2-deoxyglucose and glucose. Complementary in vivo measurements were performed to reveal SSDs response to hyperoxia and normoxia after ischemic stroke. ResultsSSD signals persisted in acute brain slices in the absence of blood flow. Inhibition of glycolysis significantly reduced SSD, while restoration of metabolic substrates partially recovered the signal. In in vivo measurements, SSD increased during hyperoxia compared to normoxia after ischemic stroke, suggesting increased oxygen-supported cellular metabolic activity. ConclusionsThese results indicate that SSD is sensitive to energy-dependent cellular processes closely tied to metabolic activity. SSD represents a previously uncharacterized, label-free in vivo optical contrast that enables assessment of cellular metabolic activity as well as vascular dynamics. This work establishes a mechanistic foundation for using SSD as a general optical marker of cellular viability in in vivo measurements.

Functional near-infrared spectroscopy (fNIRS) is a portable, motion-tolerant neuroimaging method particularly well suited for developmental and naturalistic research. To evaluate the utility of fNIRS for studying individual differences and longitudinal changes, we measured activation and functional connectivity during a relational reasoning task in young adults (N = 73). We sought to (1) establish whether fNIRS captures frontoparietal activation patterns consistent with prior fMRI studies using similar paradigms, (2) assess the effect of the amount of data (number of task blocks) on signal strength and precision, (3) assess the paradigms measurement properties in the form of intra- and interindividual stability of activation and functional connectivity within and across testing sessions, and (4) examine whether grouping channels into anatomical regions of interest (ROIs) conferred benefits to the above. We observed robust task-evoked activation across lateral prefrontal and parietal cortices, with effect sizes on par with prior fMRI studies. Generally, we observed diminishing returns in effect size and measurement precision beyond [~]7 minutes. Internal consistency and test-retest reliability varied across metrics; while they were very low for a specific task contrast, they were extremely high for functional connectivity, confirming the robustness of channel- and ROI-level connectivity as a stable marker of functional architecture. Exploratory analyses supported prior observations of lower signal quality in participants with darker skin tones and hair, underscoring the need for inclusive methodological strategies. Together, these findings highlight key design considerations for optimizing longitudinal and individual-differences research on higher-level cognition, particularly in diverse and developmentally variable populations. HighlightsO_LIWe measured within- and between-session reliability of fNIRS metrics C_LIO_LICollecting more data yielded diminishing returns in effect size and precision C_LIO_LIThere were tradeoffs to aggregating channel data into regions of interest C_LIO_LIGeneral task activation was more reliable than a specific task contrast C_LIO_LIFunctional connectivity showed extremely high test-retest reliability C_LI

Long-Stokes-shift fluorophores enable high sensitivity and multiplexed imaging with single-wavelength excitation. Under single-photon illumination ATTO 490LS exhibits a 165-nm Stokes shift, but its two-photon properties remain uncharacterised. Emission and excitation spectral analyses of ATTO 490LS in ex vivo Drosophila melanogaster brains identified two-photon excitation sensitivity at 940 nm, with peak emission at 640 nm. We demonstrate successful duplexed imaging of ATTO 490LS alongside Alexa Fluor 488 using a single 920-nm fibre laser and dual photomultiplier tubes, enabling distinct measurement of red and green fluorescence signals. These findings establish ATTO 490LS as suitable for multicolour two-photon microscopy with single-laser systems.

SignificanceSelecting the appropriate motion artefact (MA) correction method for functional near-infrared spectroscopy (fNIRS) data is quite challenging, particularly in light of the need for standardised practice, replication, and transparency in the field. A clear framework for making measurable and replicable decisions is therefore essential. AimThis paper proposes a guide based on an open-source data quality assessment tool (QT-NIRS) that enables a transparent and evidence-driven choice of MA correction method. ApproachWe present the guide in two approaches: a simplified version that is easy to run for beginners, and an advanced version providing more informative output at the cost of additional computations and minor changes to the original QT-NIRS code. Due to its high flexibility and within-subject nature, the method is applicable across samples with varied characteristics. ResultsWe applied the guide to two challenging datasets from 60 British preschoolers (mean age = 3.94 years, SD = 0.49) and 39 South African school children (mean age = 12.00 years, SD = 0.51). Both simplified and advanced approaches supported similar MA correction methods. ConclusionsWhile both approaches can be used interchangeably, we recommend the advanced approach when possible due to its more informative and straightforward output, and advise caution when using the simplified version.

Previous studies have shown the hemodynamic response to transcranial photobiomodulation (tPBM) in localized cortical regions during and after forehead irradiation. However, it is unclear if tPBM can reach deeper regions such as subcortical tissue. It is also unclear whether the manner of regional neurovascular coupling predominantly studied using tPBM extends to all brain regions. As an alternative to forehead delivery, intranasal PBM (iPBM) uses the pathway of the cribriform plate, which is thin and directly leads to the orbitofrontal cortex, rather than the prefrontal cortex in the case of forehead PBM. Thus, it is possible that iPBM can stimulate the brain more efficiently (i.e. with less power). In this study, healthy young adults underwent different iPBM protocols differing in wavelength, frequency and irradiance. We utilized functional magnetic resonance imaging (fMRI) to quantify regional blood oxygenation (BOLD) and perfusion. We further model the neurovascular interactions underlying the fMRI response. We uncovered three distinct temporal signatures, varying by brain region. Specifically, a significant response in the thalamus was observed, with a time-locked BOLD response. Overall, iPBM was found to be associated with much higher efficiency at eliciting BOLD fMRI responses than its forehead (tPBM) counterpart. Lastly, in addition to the expected dose dependence, there were extensive sex differences in the fMRI response to iPBM, surpassing those observed for tPBM. Collectively, these findings highlight the feasibility and efficacy of iPBM and establish a foundation for personalizing PBM protocols for optimal outcomes.

Functional near infrared spectroscopy (fNIRS) is increasingly used in hearing and communication research, with advantages such as robustness to movement artifacts, improved spatial resolution, and flexibility of contexts in which it can be applied. At the same time, the field is progressively moving towards more continuous, naturalistic listening paradigms resulting in the widespread adoption of speech tracking analyses such as temporal response functions (TRFs) in electroencephalography (EEG) and magnetoencephalography (MEG) studies. However, it remains unclear whether these analyses can be applied to slower haemodynamic signals measured by fNIRS. In the present study, we investigated whether a TRF framework can similarly be applied to fNIRS data recorded during continuous speech perception. Eight participants listened to speech simultaneously while fNIRS signals were acquired in a hyperscanning setup. Speech features were regressed onto the haemodynamic responses to test the feasibility and interpretability of fNIRS-based TRFs. Prediction correlations between observed and modelled fNIRS signals across speech features were higher than those typically reported for EEG- and comparable to those reported for MEG-TRF studies. Moreover, these correlations did not overlap with a null distribution generated from triallJmismatched fNIRS data, confirming statistical significance and were slightly greater than those obtained from a conventional GLM approach. Our findings support that TRF estimation method can yield meaningful and statistically significant responses from fNIRS data. HighlightsO_LITRF modelling can be meaningfully applied to fNIRS data acquired during speech listening tasks. C_LIO_LIPrediction correlations between actual and modelled fNIRS signals were above chance level, with values comparable to previous EEG/MEG studies. C_LIO_LITRFs explained more fNIRS variance than a conventional GLM approach. C_LI

A functional blood-brain barrier (BBB) is essential for the central nervous system (CNS) homeostasis and its disruption is an early event in acute brain injury and chronic neurodegeneration. Hypoxia triggers BBB breakdown, promoting endothelial dysfunction, oxidative stress, metabolic dysregulation and thrombo-inflammatory responses that compromise barrier integrity. However, strategies that directly restore BBB function remain limited. Here, we investigated whether photobiomodulation (PBM), a non-invasive light therapy, can rescue BBB dysfunction following acute hypoxic stress. Using a multicellular in vitro BBB model comprising immortalised human brain microvascular endothelial cells, pericytes and astrocytes, we induced hypoxic injury (6 h, 1% O2) and applied three PBM treatments during recovery. Hypoxia significantly reduced transendothelial electrical resistance (TEER), whereas PBM restored barrier function in endothelial monocultures and tri-cultures. Endothelial cells showed the strongest hypoxic response, with increased hypoxia-inducible factor-1, plasminogen activator inhibitor-1 and von Willebrand factor (vWF), all attenuated by PBM. Importantly, siRNA-mediated knockdown of vWF partially recapitulated PBM-induced barrier rescue, identifying endothelial vWF as a mediator of recovery. PBM also reduced reactive oxygen species in hypoxic astrocytes and pericytes, indicating coordinated multicellular modulation. These findings demonstrate that PBM restores BBB integrity after hypoxic insult by modulating endothelial thrombo-inflammatory signalling while reducing oxidative stress in glial cells. Rather than acting as a general cytoprotective stimulus, PBM engages defined molecular pathways linked to endothelial activation. This work establishes a mechanistically informed platform for studying BBB repair and supports PBM as a targeted strategy to protect vascular integrity in hypoxia-associated neurological disorders. Key points summaryO_LIHypoxia is a major driver of blood-brain barrier (BBB) dysfunction, yet there are currently no targeted therapies that directly restore barrier integrity. C_LIO_LIPhotobiomodulation (PBM) is a non-invasive low-level light intervention known to facilitate mitochondrial function and cellular stress responses. C_LIO_LIIn a human in vitro BBB model, repeated PBM treatment restored transendothelial electrical resistance (TEER) 24 and 48 hours after hypoxic injury, with endothelial rescue linked to downregulation of von Willebrand factor (vWF). C_LIO_LIPBM modulated oxidative stress, hypoxia signalling, and thrombo-inflammatory pathways across endothelial cells, astrocytes, and pericytes. C_LIO_LIThese findings support light-driven modulation of endothelial signalling as a potential strategy to restore BBB integrity in hypoxia-associated neurological conditions. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/706027v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@987f1corg.highwire.dtl.DTLVardef@1c12d86org.highwire.dtl.DTLVardef@193ea8dorg.highwire.dtl.DTLVardef@bf8d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glycated hemoglobin (HbA1c) is a central biomarker for long-term glycemic control and diabetes management, traditionally quantified using laboratory-intensive chromatographic or immunochemical assays. As the global burden of diabetes continues to rise, there is growing interest in alternative, scalable approaches capable of rapid biochemical assessment. Fourier-transform infrared (FTIR) spectroscopy offers a reagent-free method that captures molecular signatures of protein glycation, but translating complex spectra into clinically interpretable HbA1c values requires robust analytical frameworks. Here, we present a complementary multi-model strategy for predicting HbA1c from FTIR spectra of whole blood. Using 685 blood samples with matched reference HbA1c measurements, we evaluated three analytically distinct yet synergistic approaches: partial least squares regression (PLSR), peak-resolved curve fitting based on pseudo-Voigt functions combined with H2O AutoML, and a convolutional neural network (CNN). PLSR and CNN models were trained on biologically informative spectral regions (800-1800 cm-{superscript 1} and 2800-3400 cm-{superscript 1}), while curve fitting focused on the fingerprint region (1000-1720 cm-{superscript 1}) to extract interpretable biochemical parameters. PLSR achieved the highest predictive accuracy (R{superscript 2} = 0.76), closely followed by the CNN (R{superscript 2} = 0.73), reflecting their ability to capture global linear and nonlinear spectral relationships. Although curve fitting yielded lower predictive performance (R{superscript 2} = 0.59), its peak-level decomposition enabled mechanistic interpretation of glycation-related changes. Explainable AI analysis using SHAP identified lipid- and protein-associated vibrations, carbohydrate-linked glycation bands, and amide-region structural features as key contributors to HbA1c prediction. Rather than treating these approaches as competing alternatives, our results demonstrate that their integration provides a more informative framework than any single model alone. By combining predictive performance with biochemical interpretability, this multi-model FTIR strategy highlights a scalable and mechanistically grounded pathway toward non-invasive HbA1c assessment and broader metabolic screening in diabetes monitoring. The code for this study is freely available at https://github.com/MelnychenkoM/ftir-hba1c-prediction.

A critical challenge in endocrine neurosurgery is intraoperative discrimination between normal pituitary tissue and pituitary neuroendocrine tumors (PitNETs). Suggesting the universal persistence of near-infrared autofluorescence (NIRAF) in endocrine organs and inspired by routine clinical use of NIRAF for parathyroid gland identification, we discovered that pituitary NIRAF can be employed for label-free transsphenoidal surgery guidance. Ex vivo confocal spectral imaging of 33 specimens identified secretory granules as the dominant long-wavelength fluorescence source and showed that normal pituitary had higher granule content than PitNETs. For the first time, we made use of the pituitary NIRAF during surgery and assessed its performance for pituitary/adenoma separation in vivo for 27 surgeries and showed near-perfect separability between pituitary and non-pituitary measurement sites with ROC-AUC of 0.98. The obtained results clearly demonstrate that the suggested method, based on the solid microscopic background, has the potential for clinical translation and paves the way for enhanced gland preservation during resection.

Red genetically encoded calcium indicators (GECIs) are important tools for live cell and in vivo imaging. However, their application in optogenetic experiments has been limited by their complex photophysics, which can yield blue-light-induced artifacts. These photophysical drawbacks arise from the fluorescent protein (FP) used to construct the GECI. To address these limitations, we engineered novel red GECIs based on photostable red FPs, including mScarlet variants. After testing multiple design topologies and screening for calcium responses, we identified a lead variant, named ScaRCaMP-1.0. ScaRCaMP-1.0 exhibits moderate Ca2+ responses ({Delta}F/F0 = -13%) relative to other red GECIs, a tradeoff that appears to have enabled remarkable blue-light photostability at power densities exceeding 200 mW/mm2. We validated the performance of ScaRCaMP-1.0 in an optogenetic regime, and in vivo via fiber photometry. Finally, guided by structural predictions, we investigated the mechanism underlying ScaRCaMP responses. A pair of lysine residues on the surface of the FP appear to be important for controlling Ca2+ responses, and mutation of one residue (K132Y) notably increased the response size ({Delta}F/F0 = -22%) without compromising blue-light photostability. We call the improved variant ScaRCaMP-2.0. Taken together, these results establish ScaRCaMP as an optogenetics-compatible red GECI and demonstrate the potential of mScarlet-based fluorophores as a basis for generating photostable red biosensors.

The human cerebral microvasculature is both essential for brain function and highly vulnerable, yet its in-vivo structure and local hemodynamics remain largely unexplored due to the lack of imaging techniques capable of resolving deep microvascular flow in humans. This limitation not only constrains fundamental neurovascular research, but poses life-altering risks during neurosurgery, where damage to small perforating arteries can have devastating neurological consequences. Specifically, the deep cerebral perforators, branching from the major trunks of the Circle of Willis, supply essential regions of the cerebral central core but remain beyond the resolution of current intraoperative imaging modalities. Here, we report a first in-human cohort study (10 patients) demonstrating the use of 4-dimensional ultrasound localization microscopy (4D-ULM) for the intraoperative visualization of cerebral microvascular anatomy and hemodynamics. In eight patients, 4D-ULM enabled volumetric mapping of deep perforators with sub-millimeter spatial resolution ([~]140 {micro}m) at depths reaching 7 cm. This approach revealed detailed flow patterns within the previously inaccessible deep vascular networks of the human brain. Our results open new opportunities for studying microvascular physiology and could enhance intraoperative decision-making by providing high-resolution hemodynamic data, paving the way for improved microsurgical precision in neurosurgical procedures.

Infrared nerve stimulation (INS) offers millisecond-scale, electrical artifact-free activation of peripheral nerves, yet rat ex vivo studies remain scarce. We present an INS setup utilizing a lens system for free-beam focus for rat sciatic nerves partially submerged in a nerve bath, making re-wetting of the tissue in existing ex vivo INS setups redundant. Excised sciatic nerve preparations were illuminated at 1470 nm, delivering pulse trains from 100 - 2000 s at 5 Hz with radiant exposures up to 25.7 J/cm2. Compound action potentials (CAPs) were recorded with tungsten hook electrodes. Nerve activity could be recorded up to 200 minutes after excision, though persistent nerve excitability remained comparable to existing ex vivo studies as a yet to be optimized parameter. Peak CAP amplitudes ranged from 3.9 - 36.9 {micro}V with latencies averaged at 3.4 ms. Activation thresholds spanned 1.75 - 13.05 J/cm2. Additionally, potential pitfalls for INS setups and ways to resolve them were investigated, with two artifact types highly relevant to ex vivo INS being identified: A photo-thermal expansion artifact and a photovoltaic artifact with thermo-capacitive coupling. The observations confirm that CAPs can be evoked via INS in an ex vivo preparation of the rat sciatic nerve. The presented platform supports 3R principles and offers a robust basis for introduction of pharmacological investigations with INS.

Polarization-resolved second harmonic generation microscopy provides structural information about non-centrosymmetric biological samples such as collagen. It involves illuminating the sample with a focused laser beam having a variable linear polarization angle and recording the second harmonic signal as a function of this angle. However, accurate linear polarization control is challenging due to ellipticity introduced by reflections from mirrors and dichroic mirrors in the optical path. Waveplate-based compensation has emerged as the standard approach to address these distortions, but its effectiveness for quantitative measurements remains incompletely characterized. Here, we attempt to fill this gap by implementing an established automated waveplate compensation method based on a rotating half-waveplate in combination with a compensating quarter-waveplate. This was done on a commercial Leica TCS SP8 MP multiphoton microscope, making various hardware improvements and carefully documenting important experimental details. Despite significant effort, we consistently observed substantial unwanted residual polarization ellipticity, with amplitudes up to 0.25, persisting under optimal waveplate configurations. Our simulation analysis provides evidence that this limitation may arise from wavelength-dependent dichroic mirror birefringence combined with the broad spectral bandwidth (10nm to 20nm full width at half maximum) of femtosecond laser pulses. While the approach investigated here can compensate a single wavelength, different spectral components within the pulse experience different phase retardations from wavelength-dependent optical elements, potentially resulting in residual ellipticity that cannot be eliminated. Our simulations qualitatively reproduced key features of the experimental observations. These findings have important implications for quantitative polarization-resolved second harmonic generation microscopy and suggest that alternative approaches, including specimen rotation or picosecond laser sources with narrower bandwidth, should be investigated for applications requiring precise polarization control. To facilitate community investigation of these effects, we provide open-source analysis code and simulation files.

Motion artifacts remain a barrier to in vivo calcium imaging in Drosophila melanogaster larvae. Here, we evaluate a multimodal immobilization approach that combines a Pluronic F-127 (PF-127) hydrogel with brief diethyl ether vapor exposure (5 minutes, 25{degrees}C) and compare it against hydrogel-only immobilization using custom MATLAB-based analysis software that performs NoRMCorre rigid motion correction. In wide-field GFP recordings at 1 Hz over approximately 60 minutes (N = 15 per group), the multimodal condition significantly reduced motion across all three core metrics after FDR correction (all q < 0.001), with large effect sizes for mean speed (Hedges g = -1.18) and median step size (g = -1.36). In a secondary analysis of the first 30 minutes, uniformly large effect sizes (|g| = 1.10-1.51) were observed, consistent with stronger initial chemical immobilization that partially wanes over the recording period. We implemented a dual-flag quality control system that distinguishes motion data reliability from ROI detection eligibility. Control calcium recordings (33.33 Hz, [~]5 minutes; N = 23) yielded 368 ROIs with a mean SNR 30.4 {+/-} 16.9 and an event rate of 0.228 {+/-} 0.113 Hz. Experimental recordings (N = 21) yielded 295 ROIs with SNR 18.0 {+/-} 10.6 and event rate 0.309 {+/-} 0.188 Hz. SNR was higher in controls (Cliffs{delta} = 0.50, p < 0.001), while event rate was modestly higher in the experimental group at the ROI level ({delta} = -0.22, p < 0.001), though this difference did not reach significance at the sample level, suggesting altered but not suppressed calcium dynamics. These results support a practical, accessible immobilization workflow for larval calcium imaging. HighlightsO_LIBrief ether + hydrogel approach reduces larval motion 85-91% vs. hydrogel alone C_LIO_LIDual-flag QC system separates motion reliability from calcium ROI eligibility C_LIO_LICalcium event rates not suppressed under multimodal immobilization C_LIO_LIComplete MATLAB pipeline for motion analysis and calcium imaging provided C_LIO_LIAccessible protocol requires only standard laboratory supplies C_LI

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca2+ channels and Ca2+ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca2+ dynamics at the soma is often performed, the imaging of Ca2+ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca2+ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca2+ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca2+ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

Longitudinal, noninvasive in vivo imaging is essential for studying brain physiology and pathological mechanisms. Advances in transcranial optical windows, including thinned-skull and optical clearing techniques, have markedly improved imaging depth and resolution when combined with multiphoton microscopy. However, their optical performance often deteriorates rapidly, and quantitative studies on long-term stability and the causes of image quality loss remain limited. In this work, we systematically investigated current transcranial window approaches using multiphoton excited fluorescence microscopy (MPEFM) and adaptive optics, examining longevity, optical aberrations, and imaging resolution. Our results reveal that progressive skull regrowth is a fundamental limitation across all window types, leading to substantial declines in signal quality and resolution for conventional MPEFM thereby reducing achievable high-resolution imaging depth. To address these challenges, we developed a localized glucocorticoid (GC) delivery strategy that significantly extends window performance for up to one month. Furthermore, we demonstrated that a GC-loaded hydrogel sealing method effectively suppressed skull regrowth while preserving optimal optical properties, offering a potential and practical route to chronic, high-fidelity transcranial imaging. These findings provide mechanistic insight into window degradation and establish a framework for sustained, long-term in vivo brain imaging.

Astrocytes are crucial mediators of diverse aspects of brain function, including energy metabolism and synapse formation and maturation. Calcium is the primary information carrier in astrocytes, reporting cellular health and activity, and can be measured using fluorescent indicators. However, this readout is not yet widely used to screen and evaluate disease models and drug candidates. Here, we adapted a simple automated calcium imaging pipeline with key output parameters that characterize changes in astrocytic calcium signaling. We compared calcium responses in mouse astrocyte monocultures and astrocyte-neuron cocultures using GFAP-driven membrane-targeted GCaMP6f, with human astrocytes differentiated from two different induced pluripotent stem-cell lines using the calcium dye Cal520-AM. Event-based analysis reported similarities and differences in mean fluorescence, amplitude, frequency, duration, and area of calcium responses. We benchmarked the pipeline using the purinergic receptor agonist ATP to increase astrocyte activity, and the ER calcium pump blocker CPA to decrease activity across all culture models. Glutamatergic and serotonergic receptor function was tested with glutamate and lysergic acid diethylamide (LSD). LSD decreased activity in mouse cocultured astrocytes, but increased activity in human astrocytes. Furthermore, the addition of human recombinant Tau oligomers, an in vitro model of Alzheimers disease pathology, decreased activity in both mouse and human astrocytes. This pipeline can be used to quickly and easily characterize effects of astrocyte-targeting compounds, effects of non-astrocyte-targeting compounds on astrocyte activity, and rescue of disease models that affect astrocyte function, in mouse and human astrocytes and astrocyte-neuron cocultures.

SignificanceMastectomy skin flap necrosis remains a major complication in implant-based breast reconstruction due to inadequate tissue blood flow. Existing diagnostic technologies are limited by shallow depth sensitivity, dye-related risks, contact requirements, and an inability to continuously assess blood flow. AimThis study aimed to translate a noncontact, dye-free, depth-sensitive speckle contrast diffuse correlation tomography (scDCT) technique to a clinically relevant porcine skin flap model for assessing flap blood flow and viability. ApproachThe scDCT system was optimized to image blood flow over seven days in four porcine skin flaps including Sham (SH), Implant (IM), Half Necrosis (HN), and Full Necrosis (FN). Measurements were compared with indocyanine green angiography (ICG-A) as a reference standard. ResultsscDCT enabled longitudinal monitoring of flap blood flow, revealing significant flow differences among flap types and over time. FN flaps consistently exhibited the most severe flow impairment, while other flap types showed partial or complete recovery over time, distinguishing nonviable from viable tissue. scDCT measurements demonstrated moderate to strong correlations with ICG-A across time points. ConclusionsThe findings support scDCT as a promising perioperative imaging modality for improving flap necrosis risk stratification and surgical decision-making, with future work focused on large-scale validation and clinical translation.

Longitudinal brain imaging is essential for understanding neural mechanisms. Here, we present a saline-free, chronic preparation for repeated neural recording in adult Drosophila over multiple days. We describe steps for mounting flies, performing manual surgery on the head cuticle without external saline, and resealing the opening to create a transparent optical window. We demonstrate the utility of this approach by tracking single-neuron spiking and neuronal calcium dynamics over 7-10 days. This protocol is potentially applicable to other insect species. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/706199v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@abeb34org.highwire.dtl.DTLVardef@deaf93org.highwire.dtl.DTLVardef@1d8fc24org.highwire.dtl.DTLVardef@91a696_HPS_FORMAT_FIGEXP M_FIG C_FIG