Neurophotonics

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.

Wide-field imaging (WFI) is a mesoscopic approach for monitoring cortex-wide activity with high temporal resolution and a broad field of view. Owing to its simple optical configuration and compatibility with chronic preparations, WFI has become an important tool in systems neuroscience and disease-model research. In this chapter, we describe practical protocols for chronic transcranial WFI in mice using two complementary optical signals: genetically encoded calcium indicators (GCaMP) and endogenous flavoprotein autofluorescence. Calcium imaging provides a robust readout of neuronal population activity, whereas flavoprotein imaging reflects mitochondrial redox dynamics and cellular metabolic demand. We detail procedures for animal preparation, skull clearing, headplate implantation, macroscope assembly, synchronized sensory stimulation, triggered image acquisition, and MATLAB-based data analysis. The analysis workflow includes {Delta}F/F normalization, reference-based signal correction, and artifact reduction, followed by trial averaging, atlas registration, and region-of-interest analysis. Because imaging is performed through the intact skull, the protocol enables repeated longitudinal measurements in the same animal over extended periods. This approach is reproducible, cost-effective, and adaptable to studies of cortical physiology and neurological disorders.

Long-term fiber photometry enables measurement of neural dynamics across hours to days, but these recordings create analytical and reproducibility challenges that are not well addressed by tools developed for short, stimulus-locked experiments. Here we present a software environment for long-term photometry analysis organized around a structured, revisitable workflow for run execution, inspection, and post-run refinement. The software separates correction retuning from downstream event reanalysis, allowing both signal correction and event-analysis settings to be revised after the initial run. We show that correction choice can substantially change the corrected signal itself and that post-run reanalysis can revise event-detection outcomes. The software also preserves tonic and phasic outputs and supports inspection of the same recording at both multiday and session-level scales. Together, these capabilities provide a practical workflow for more interpretable, revisitable, and reproducible analysis of long-term photometry recordings.

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.

Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, most red fluorescent proteins require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant (LSSmScarlet3) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show that 920 nm elicits robust in vitro and in vivo fluorescence. Channel crosstalk is minimal. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.

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.

Myelin is a highly complex membranous structure wrapped around axons by oligodendrocytes or Schwann cells in the central and peripheral nervous system, respectively. Fluorescent labeling is widely used to study the structure and dynamics of myelin. Combining structural with functional imaging requires labeling of myelin with red fluorescence, as many functional sensors, including Ca2+ indicators and genetically encoded metabolite sensors, fluoresce in the green spectral range. However, in vivo tools enabling red fluorescent labeling of myelinating cells and their myelin sheaths remain limited. Here, we generated a set of seven transgenic mouse lines expressing a membrane-targeted variant of the red fluorescent protein tdTomato in myelinating oligodendrocytes and Schwann cells throughout the nervous system. The mouse lines provide a variety of expression patterns ranging from wide-spread labeling of myelin to a rather sparse expression, the latter enabling visualization of individual oligodendrocytes and their associated myelin sheaths. In the peripheral nervous system, the pattern of fluorescence in sciatic nerves indicates predominant localization of tdTomato to non-compact myelin compartments including the inner and outer tongues, paranodal loops and Schmidt-Lanterman incisures. In summary, our work provides a set of novel mouse lines with myelin labeled by red fluorescence, which are compatible with diverse imaging modalities in the green spectral range enabling integrated structural and functional imaging. Main PointsO_LITransgenic mouse lines expressing membrane-targeted tdTomato in myelin enable imaging of myelin in the red spectral range C_LIO_LIDistinct expression patterns range from wide-spread labeling to sparse single-cell resolution, supporting diverse imaging applications C_LI

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 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.

When hearing fails, cochlear implants (CIs) partially restore auditory perception. Yet, poor coding of spectral information remains a bottleneck as each electrode broadly activates the auditory nerve. As light can be more conveniently confined, optical (o)CIs present a promising alternative. Here, we combined expression of the potent channelrhodopsin ChReef in spiral ganglion neurons (SGNs) and oCIs based on 5-10 green LED in gerbils. We characterized the oCI encoding of intensity and spectral information by ChReef-SGNs using recordings from the central nucleus of the inferior colliculus (ICC). ChReef aligned light sensitivity of SGNs well with the radiant fluxes provided by individual LEDs: ICC-activity had thresholds <200 nJ and reached a maximum close to that achieved with 46 dB tones. Multichannel oCIs enabled tonotopically ordered and spectrally distinct stimulation indistinguishable from acoustic stimulation for up to moderate activity levels. Some LEDs elicited >1 spectral peaks for stronger intensities. Representational Similarity Analysis and Linear Discriminant Analysis of ICC activity indicated improved channel discriminability of optical over electrical stimulation. In summary, {micro}J oCI stimulation achieves near-physiological spectral resolution. The Paper ExplainedO_ST_ABSProblemC_ST_ABSElectrical cochlear implants (eCIs) partially restore speech comprehension in most of >1 million otherwise deaf users, who still face challenges hearing in daily situations. This is primarily due to poor spectral selectivity of electrical sound encoding. Spatially more confined optogenetic activation of the auditory nerve by optical cochlear implants (oCI) promises to overcome this limitation. However, a thorough characterization of bionic coding of sound information by multichannel oCI is needed to evaluate the potential for improved hearing restoration. ResultsHere, we combine the potent channelrhodopsin ChReef and 10-channel oCI based on green LEDs in gerbils and characterize their utility for encoding of spectral and intensity information by multielectrode array recordings from the midbrain. ChReef enabled activation of the auditory pathway with nano-joule thresholds and up to high levels of midbrain activity with low {micro}J radiant energy. The cochlear spread of excitation and channel discriminability for low to medium activity levels were close to what we observed with acoustic stimulation. ImpactOur work demonstrates great potential of multichannel optogenetic stimulation for encoding sound frequency information.

A central challenge in opsin engineering is identifying mutations that reliably improve desired functional properties, a task made difficult by the enormous mutation space and limited throughput of electrophysiological screening. Improving opsin properties such as photocurrent amplitude and light sensitivity have the potential to broaden the use of opsins to low-light and deep-tissue applications. With this goal, we applied zero-shot protein language models (ESM-1b/1v) to recommend ChrimsonR mutations and experimentally validated all 17 of these variants using whole-cell patch clamp electrophysiology (n=6 cells per mutation). Despite many mutations reducing function, protein language models identified both known functional residues and unconventional substitutions that produced large functional gains and synergized with K176R to improve kinetics. Two mutations, E300G and E300P, increased sustained photocurrents from 66 pA (control) to 305 pA and 255 pA at 635 nm, reduced EC50 at 575 nm from 0.19 mW to 0.07 mW, and altered kinetics ({tau}off increased from 0.06 s up to 0.40 s). Our results suggest that protein language models, even without task-specific training, can be used alongside electrophysiological measurements as a strategy for screening opsins for enhanced photocurrent.

Understanding neural correlates of brain function in neuroscience now largely involves detecting and analyzing transient signals from fluorescent sensors. Imaging technologies such as confocal and two-photon microscopy, along with onboard miniscopes, enable the visualization of neural activities and capture dynamic signals both ex vivo and in vivo. This includes monitoring Ca2+ transients via the expression of genetically encoded sensors such as GCaMP in specific brain cells. Additionally, the advent of GPCR-based neurotransmitter sensors allows for imaging the release of neurotransmitters including glutamate and GABA, as well as neuromodulators such as dopamine or noradrenaline. These approaches however generate large, high-dimensional, spatiotemporally complex datasets, presenting significant challenges for signal detection and analysis. To overcome these challenges, we developed a versatile pipeline of Dynamic Extraction and Tracking of Emitted Cellular Transients (DETECT), which combines background denoising, object segmentation, and multi-object tracking. Our user-friendly, Python-based GUI offers a low-resource platform for efficient data analysis. Validated across various imaging modalities and biological models, DETECT provides a robust and comprehensive solution for analyzing complex imaging datasets in neuroscience research.

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.

1.We report a repetition-controllable gain-managed nonlinear fiber amplifier (GMNA) that delivers near-infrared 50-fs pulses with pulse energies up to 150 nJ and a widely tunable repetition rate from 1-20 MHz, while maintaining stable pulse quality across the full range. Using this source, we demonstrate label-free multiphoton imaging--including metabolic autofluorescence (2PF/3PF), second/third-harmonic generation, and Simultaneous Label-free Autofluorescence Multiharmonic (SLAM) microscopy imaging--across live cells, human lung spheroids, and hard tissues. We further assess the impact of laser repetition rate on photodamage at fixed pulse energy, supported by preliminary measurements indicating lower damage at lower repetition rate. Collectively, the compact architecture and repetition-rate agility of the GMNA enable real-time optimization of imaging speed, depth, and sample safety for advanced biological microscopy.

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

Dark adaptation is the essential process that restores visual sensitivity following exposure to bright light, yet the underlying mechanisms remain incompletely understood. Here, we propose a method for assessing dark adaptation in cones using optoretinography (ORG) based on adaptive optics optical coherence tomography (AO-OCT). ORG quantifies cone functional response by monitoring nano-scale changes in the cones outer segment occurring over hundreds of milliseconds after visible stimulation. This method consists of sequential measurements of stimulus-evoked cone responses over the course of minutes of dark adaptation. Each response captures optical path length changes in single photoreceptor outer segments over milliseconds during a multi-minute recovery period following a strong photopigment bleach. We parameterized cone ORG responses and proposed an exponential model linking ORG dynamics to pigment regeneration. Parameters of the ORG response exhibited exponential decay behavior during dark adaptation, and were thus fit with exponential functions and quantified by the resulting decay parameter{tau} . Parameters capturing the amplitude of the ORG responses recovered more slowly than those capturing temporal dynamics of the responses. This difference is consistent with distinct contributions from photopigment regeneration and downstream phototransduction processes. Recovery speed varied by two-to threefold among three normal subjects, suggesting substantial inter-subject physiological diversity. Processes within the cone, including pigment regeneration, are thought to underlie the gains in photopic visual sensitivity that occur in the dark. These findings highlight ORG as an objective and sensitive assay of those cellular mechanisms. While the ORG itself has shown promise as a biomarker of the health of the photoreceptor response to light, the results of this study show that it may also be useful for probing the health of the intra- and intercellular homeostatic mechanisms that support it.

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.

Opfically pumped magnetometer-based magnetoencephalography (OPM-MEG) has recently emerged as a powerful neuroimaging approach in cognifive neuroscience, extending beyond the limitafions of convenfional cryogenic systems with greater experimental flexibility and wearable recording. Despite these advantages, standardised data analysis frameworks specifically tailored to OPM technology are sfill lacking, leading to variability in processing choices and reduced reproducibility across laboratories and hardware plafforms. We introduce OPM-FLUX, a comprehensive and fully documented end-to-end analysis pipeline developed for OPM-MEG data. The pipeline defines a clear sequence of preprocessing, noise suppression, arfifact handling, spectral analysis, evoked response analysis along with recommended parameter seftings. It also includes source reconstrucfion to idenfify where in the brain the signals originate. In addifion, OPM-FLUX supports mulfivariate paftern analysis (MVPA), enabling fime-resolved decoding of cognifive processes from sensor level data. OPM-FLUX is implemented in MNE-Python and distributed as interacfive Jupyter Notebooks that combine executable code with detailed methodological explanafions and graphical outputs. The pipeline further provides standardized reporfing templates and a data acquisifion Standard Operafing Procedure to facilitate preregistrafion, consistent documentafion, and standard pracfices across research sites. The workflow is demonstrated using openly available datasets acquired from both Cerca/QuSpin and FieldLine OPM systems during a visuospafial aftenfion paradigm that modulates alpha, beta, and gamma oscillafions and elicits event-related responses. By supporfing mulfiple OPM plafforms and promofing consistent methodological choices, OPM-FLUX enhances transparency, comparability, and replicafion in OPM-MEG research. The pipeline also serves as an educafional resource for students and researchers entering the field and is designed to evolve alongside ongoing technological and methodological advances in OPM-based brain imaging.

The photon measurement density function (PMDF) plays a fundamental role in both pre-experimental optode arrangement and post-experimental data analysis in functional near-infrared spectroscopy (fNIRS). Conventionally, PMDFs are derived from structural MR images through tissue segmentation and photon propagation modeling (PPM), which are computationally demanding and time-consuming, thereby limiting their practical use. In this study, we propose a novel deep learning-based framework to estimate PMDFs directly from MR images and channel configurations. The proposed method supports flexible source-detector distances and eliminates the need for explicit tissue segmentation and repeated photon simulations. Specifically, a convolutional neural network is trained to predict photon fluence distributions, from which PMDFs are subsequently derived using the adjoint formulation. The trained model is evaluated on channels placed in both trained and unseen scalp regions across commonly used source-detector distances. The results demonstrate that the proposed method achieves PMDF estimations comparable to those obtained from PPM. Overall, this approach significantly reduces computational cost and has the potential to facilitate broader adoption of PMDF-based methods in the fNIRS community.

Microglia represent the immune component of the central nervous system (CNS) that displays dynamic responses to injury and disease. Across the developing and mature CNS, microglia emerge as immunocompetent cells that continuously survey their surroundings to maintain tissue homeostasis and respond to threats. There remains a gap in 3D in vitro models that contain microglia and can provide both developmental and mature functional hallmarks. Using a 3D neural multicellular model, cortical microtissues, derived from primary rat cortical cells, we conducted live imaging to monitor microglia dynamics from early, middle, and late stage microtissue maturation. We optimized a within-micromold imaging approach that allows for live microglia imaging without removing microtissues from their culturing environment. We confirm that microglia exhibit baseline surveillance characterized by relatively stationary somas and highly dynamic cell processes that continuously extend and retract. Following proinflammatory challenges, microglia engulf lipopolysaccharide particles, accompanied by dynamic shifts in motility patterns; and rapidly respond to laser-induced tissue damage through process extension, whole-cell displacement, and local recruitment. Lastly, we show that microtissue age in culture strongly influences both baseline and directed motility profiles. Collectively, these studies demonstrate that within a 3D microenvironment, microglia exhibit pronounced changes in morphology, surveillance area, motility, and injury response across microtissue maturation. Microtissues can serve as a valuable in vitro platform for both microglia developmental studies and investigations of brain inflammation related to CNS injuries, infections, and diseases.