Hanafy AS, Steinlein P, Pitsch J, Silva MH, Vana N, Becker AJ, Graham ME, Schoch S, Lamprecht A, Dietrich D. Subcellular analysis of blood-brain barrier function by micro-impalement of vessels in acute brain slices. Nat Commun 2023;14(1):481. doi: 10.1038/s41467-023-36070-6.

 

Background: The blood–brain barrier (BBB) regulates molecular exchange between blood and brain and changes dynamically with physiological and disease states. Technical limitations in maintaining tissue integrity and imaging resolution have hindered detailed study of BBB transport. This study aimed to overcome these challenges by creating a high-resolution model that enables direct visualization and quantification of BBB permeability and cellular mechanisms in real time.

 

Hypothesis: This study hypothesized that direct micro-impalement and perfusion of brain capillaries in acute slices, combined with live fluorescence imaging, would enable precise, subcellular analysis of BBB permeability, transporter function and structural integrity under both physiological and pathological conditions.

 

Methods: The authors prepared acute brain slices from mouse and human hippocampus and micro-perfused fluorescent tracers and dyes into individual vessels. Imaging was conducted with two-photon microscopy using ScanImage. Endothelial cell morphology, tracer diffusion and transporter activity were assessed using fluorescent markers including TMR, FM1-43, calcein, rhodamine123 and BSA-Alexa488. Proteomic analyses quantified transporter proteins in pilocarpine-treated animals.

 

Results: Perfusion of tracers showed that small molecules like 7-hydroxycoumarin diffused beyond vessel walls, while larger tracers remained intravascular. FM1-43 crossed endothelial junctions by membrane diffusion. ABC transporters actively extruded rhodamine123 and calcein, effects reversed by inhibitors. Chemical (DMSO, mannitol) and epileptic conditions caused selective leakage to small but not large molecules, indicating partial BBB compromise.

 

Conclusions: This study established a reproducible live-slice assay for quantifying BBB function at subcellular resolution, demonstrating active transporter-mediated exclusion, differential permeability by molecular size and localized barrier disruption under pathological conditions.

Bar-Noam AS, Farah N, Shoham S. Correction-free remotely scanned two-photon in vivo mouse retinal imaging. Light Sci Appl 2016;5(1):e16007. doi: 10.1038/lsa.2016.7.

 

Background: Non-invasive fluorescence imaging of the mouse retina is essential for studying retinal structure and function in health and disease. While two-photon microscopy provides optical sectioning and reduced phototoxicity, its in vivo use in mice has been limited by the need for adaptive optics to correct ocular aberrations. Overcoming these limitations would allow simpler and more accessible retinal imaging.

 

Hypothesis: This study hypothesized that model-based optical design combined with remote focusing could enable high-resolution, correction-free two-photon imaging of the mouse retina through the intact pupil.

 

Methods: The authors developed a two-photon imaging setup that used a tunable infrared laser and an electronically tunable lens combined with offset optics to scan axially without corneal contact. Optical performance was modeled analytically and with ray-tracing software. Mouse retinal imaging was performed using fluorescein and genetically encoded calcium indicators. Image acquisition was conducted with ScanImage, and image analysis was performed using standard image processing tools.

 

Results: The optical model accurately predicted in vivo focal shifts and resolution. The system generated high-quality, optically sectioned three-dimensional angiograms and cellular-resolution images of GCaMP-expressing retinal cells. Functional calcium imaging revealed robust, repeatable light-evoked responses in retinal ganglion cells without apparent retinal damage across repeated sessions.

 

Conclusions: This study demonstrated that a simple, adaptive-optics–free two-photon imaging system can achieve correction-free, cellular-resolution in vivo retinal imaging and functional calcium recordings, providing a practical and accessible baseline for future multimodal retinal imaging applications.

Bakker GJ, Weischer S, Ferrer Ortas J, Heidelin J, Andresen V, Beutler M, Beaurepaire E, Friedl P. Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy. Elife 2022;11:e63776. doi: 10.7554/eLife.63776.

 

Background: Intravital microscopy enables visualization of live-cell dynamics within intact tissues, but its penetration depth is limited by light scattering and absorption. Conventional two-photon (2P) microscopy typically reaches only several hundred micrometers, restricting access to deeper, more heterogeneous structures such as tumors or bone. Recent advances using three-photon (3P) excitation in the infrared spectrum have achieved greater depths in brain tissue, but their broader applicability to complex, strongly scattering tissues has not been fully characterized.

 

Hypothesis: This study hypothesized that high pulse energy, low repetition rate infrared excitation at 1300 and 1700 nm would enable deeper, multiparameter intravital imaging in dense tumor and bone tissue, while maintaining non-toxic conditions compatible with live imaging.

Methods: The authors implemented single-beam 3- and 4-photon microscopy using a laser source providing sub-100 fs pulses at 1 MHz and excitation wavelengths of 1300 and 1650 nm. Images were acquired and system control performed with ScanImage. Fluorescent tumor xenografts, bone and brain tissues were analyzed for imaging depth, signal-to-noise ratio and resolution. Phototoxicity thresholds were determined using Ca²⁺ sensors and viability markers to define safe excitation conditions.

 

Results: High infrared excitation generated simultaneous 3- and 4-photon fluorescence and harmonic signals in vivo, providing approximately twofold deeper imaging in tumors and bone compared to 2P microscopy. Effective attenuation length and resolution remained stable beyond 400 μm, and safe operation was confirmed below 8.4 nJ (1300 nm) and 12 nJ (1650 nm) pulse energies without detectable tissue damage.

 

Conclusions: High-energy, low-repetition infrared excitation combined with ScanImage-controlled multiphoton microscopy enables non-invasive, deep-tissue imaging with enhanced resolution and spectral range, advancing intravital studies of complex biological microenvironments.

Wang K, Pan Y, Tong S, Liang H, Qiu P. Deep-skin multiphoton microscopy of lymphatic vessels excited at the 1700-nm window in vivo. Biomed Opt Express 2021;12(10):6474-6484. doi: 10.1364/BOE.437482.

 

Background: Lymphatic vessels maintain fluid balance and immune function, but imaging them in skin is limited by tissue scattering. Multiphoton microscopy (MPM) provides high resolution and optical sectioning, yet shorter excitation wavelengths restrict depth. Using the 1700-nm excitation window reduces scattering and allows deeper, clearer imaging of biological structures.

 

Hypothesis: This study hypothesized that excitation of indocyanine green (ICG)–labeled lymphatic vessels at the 1700-nm window using multiphoton microscopy would enable high-resolution, in vivo imaging of both the structural and dynamic properties of deep lymphatic vessels in mouse skin.

 

Methods: The authors employed a laser scanning microscope equipped with galvo mirrors, photomultiplier tubes and a water-immersion objective lens to perform two-photon fluorescence (2PF), second-harmonic generation (SHG) and third-harmonic generation (THG) imaging in mouse hindlimb skin. ICG was injected to label lymphatic vessels, and quantum dots were used to distinguish blood vessels. Image acquisition and processing were performed using ScanImage. Noninvasive and exposed-skin preparations were imaged in vivo under isoflurane anesthesia.

 

Results: Noninvasive 2PF imaging resolved lymphatic vessels up to 300 µm below the skin surface, visualizing vessel morphology and contraction dynamics at an average frequency of 0.8 min^-1. In exposed-skin preparations, THG imaging revealed vessel walls and lymphatic valves, whose opening and closing were recorded in real time. Additionally, micrometer-sized particles flowing and transiently trapped near valves were visualized. Signal-to-background ratios reached up to 197:1, confirming image quality and depth penetration.

 

Conclusions: MPM excited at the 1700-nm window enables noninvasive, deep-tissue visualization of lymphatic vessels and their dynamics in vivo, providing a powerful tool for studying lymphatic physiology and pathology with subcellular resolution.

Heiser H, Kiessler F, Roggenbach A, Ibanez V, Wieckhorst M, Helmchen F, Gjorgjieva J, Wahl AS. Brain-wide microstrokes affect the stability of memory circuits in the hippocampus. Nat Commun 2025;16(1):3462. doi: 10.1038/s41467-025-58688-4.

 

Background: Small-vessel disease–related microstrokes are a major cause of cognitive decline and vascular dementia. The hippocampus, central to spatial and episodic memory, is particularly vulnerable to microvascular insults. However, how such microscopic lesions disrupt hippocampal network stability and memory encoding over time remains unclear.

 

Hypothesis: This study hypothesized that distributed microstrokes impair spatial memory by destabilizing hippocampal place cell networks, and that preservation of stable spatial coding predicts recovery of cognitive function.

 

Methods: The authors used chronic two-photon calcium imaging to repeatedly record the same hippocampal CA1 neurons in head-fixed mice navigating a virtual reality corridor. Microstrokes were induced by unilateral intra-arterial microsphere injections. Imaging was performed with a galvo-resonant two-photon system controlled by ScanImage. Neural data were preprocessed with automated motion correction, and neuronal activity was classified into stable, unstable or non-coding cells. Bayesian decoding and correlation analyses assessed network stability and spatial coding precision.

 

Results: Microstrokes significantly reduced task performance, mean firing rates and within-session stability of place cells without affecting motor behavior. The loss of stable place cells correlated with lesion load and chronic spatial memory deficits. Mice that recovered behaviorally showed restoration of stable place cells and improved decoder accuracy in later sessions. Recovery animals displayed increased spatial synchronization near reward zones, indicating functional reorganization of salient locations.

 

Conclusions: Microstrokes disrupt hippocampal spatial coding and network stability, leading to memory loss. Maintenance or reestablishment of stable, synchronously active place cells supports functional recovery after microvascular brain injury.

 

 

Vallentin D, Kosche G, Lipkind D, Long MA. Neural circuits. Inhibition protects acquired song segments during vocal learning in zebra finches. Science 2016;351(6270):267-271. doi: 10.1126/science.aad3023.

 

Background: Vocal imitation learning requires that sensory information from a tutor be integrated into the motor circuits responsible for song production. In zebra finches, this process involves thescain forebrain nucleus HVC, which connects auditory inputs with premotor output neurons. While juveniles show strong tutor song–evoked activity in HVC during learning, the mechanisms by which this responsiveness changes with development and learning remain unclear.

 

Hypothesis: This study hypothesized that inhibitory synaptic activity within the HVC circuit strengthens with learning and functions to suppress sensory responses in premotor neurons for song elements that have already been mastered.

 

Methods: The authors performed electrophysiological recordings in awake juvenile and adult male zebra finches. Birds were reared under controlled tutoring conditions, and recordings targeted HVC neurons projecting to the robust nucleus of the arcopallium. Sharp intracellular, juxtacellular and two-photon targeted whole-cell recordings were obtained using a customized moveable-objective two-photon microscope, a laser tuned to 800 nm, and images were captured with ScanImage. The authors used an oil-based pressure injection system, motorized micromanipulator and an amplifier for voltage-clamp recordings. In some adults, a GABA_A antagonist (gabazine) was locally applied to test the role of inhibition.

 

Results: Tutor song playback evoked precise spiking in HVC premotor neurons of juveniles but not adults. Blocking inhibition reinstated these responses in adults, revealing that synaptic inhibition suppresses sensory inputs after learning. The strength, frequency and temporal precision of inhibitory currents correlated with each bird’s similarity to the tutor song rather than age. During artificial tutoring, inhibition selectively targeted learned syllables, while unlearned segments remained less inhibited.

 

Conclusions: These findings show that learning-dependent inhibition in HVC protects acquired song elements from further modification, thereby enabling sequential mastery of complex motor sequences.

Cox J, Pinto L, Dan Y. Calcium imaging of sleep-wake related neuronal activity in the dorsal pons. Nat Commun 2016;7:10763. doi: 10.1038/ncomms10763.

 

Background: The dorsal pons has long been recognized as critical for generating rapid eye movement (REM) sleep, but the underlying circuitry and cell-type-specific mechanisms remain unclear. Distinct neuronal populations, including glutamatergic and GABAergic cells, display state-dependent activity across the sleep–wake cycle, yet their spatial organization and specific contributions to REM and wakefulness have not been well defined.

Hypothesis: This study hypothesized that distinct subpopulations of glutamatergic and GABAergic neurons in the dorsal pons exhibit characteristic and spatially organized activity patterns that correlate with specific behavioral states such as REM sleep and wakefulness.

 

Methods: The authors performed cell-type-specific calcium imaging in freely moving mice expressing GCaMP6s in glutamatergic or GABAergic neurons of the dorsal pons. Activity was recorded through an implanted gradient refractive index (GRIN) lens coupled to a miniaturized fluorescence microscope and analyzed across natural sleep–wake transitions. Two-photon imaging was conducted using a movable objective microscope controlled by ScanImage to confirm signal specificity.

 

Results: GABAergic neurons were significantly modulated by brain state and mostly active during wakefulness, with smaller subsets active during REM sleep. In contrast, most glutamatergic neurons were maximally active during REM sleep, showing increased activity at transitions into REM and decreased activity when awakening. These REM-active glutamatergic neurons were preferentially located medially, whereas wake-active neurons were more lateral.

 

Conclusions: This study concluded that dorsal pontine glutamatergic and GABAergic neurons display distinct and spatially organized sleep–wake activity patterns, suggesting that specific subpopulations within this region contribute differentially to REM sleep and wakefulness regulation.

Yang W, Miller JE, Carrillo-Reid L, Pnevmatikakis E, Paninski L, Yuste R, Peterka DS. Simultaneous multi-plane imaging of neural circuits. Neuron 2016;89(2):269-284. doi: 10.1016/j.neuron.2015.12.012.

 

Background: Understanding brain function requires recording neuronal activity across multiple cortical layers simultaneously. Conventional two-photon microscopy relies on sequential scanning, limiting temporal resolution and volumetric coverage. To overcome these constraints, the authors developed a holographic two-photon imaging system enabling simultaneous multi-plane imaging for functional mapping of neuronal populations across cortical depths with single-cell precision.

 

Hypothesis: This study hypothesized that a holographically multiplexed two-photon microscope, combined with computational signal extraction, allows simultaneous high-speed calcium imaging across several cortical planes while maintaining accuracy and spatial resolution.

 

Methods: The authors modified a two-photon microscope by integrating a spatial light modulator (SLM) into the excitation path of a galvanometer-based scanner. Imaging was performed in awake mice expressing GCaMP6f in the visual cortex. The microscope was operated and data were acquired using ScanImage, which synchronized laser modulation and multi-plane scanning. Fluorescence signals were extracted using a constrained nonnegative matrix factorization (CNMF) algorithm to separate overlapping sources. The SLM enabled flexible focusing of multiple beamlets without moving the objective, permitting simultaneous imaging of laterally or axially displaced cortical regions.

 

Results: The system achieved dual- and triple-plane imaging of neuronal populations up to 500 µm deep. CNMF extraction improved signal-to-noise ratio by about 13% over traditional methods. Neurons imaged in different planes showed highly correlated dynamics and preserved orientation selectivity.

 

Conclusions: Combining holographic multiplexing with ScanImage-controlled two-photon microscopy enables fast, simultaneous multi-plane imaging with high fidelity, providing a versatile tool for studying three-dimensional neural circuit dynamics in vivo.

Zhao S, Hebert E, Gruzdeva A, Mahishi D, Takahashi H, Lee S, Hao YA, Lin MZ, Yapici N, Xu C. Deep two-photon voltage imaging with adaptive excitation. Res Sq [Preprint] 2024 Dec 13:rs.3.rs-5434919. doi: 10.21203/rs.3.rs-5434919/v1.

 

Background: Optical monitoring of neuronal voltage dynamics enables the study of fast neural activity with subcellular resolution. However, two-photon microscopy (2PM) suffers from depth and speed limitations due to tissue heating and reduced photon flux. Existing methods either image superficial neurons or single cells in deeper layers, often requiring complex beam multiplexing or high laser powers that risk thermal damage.

 

Hypothesis: This study hypothesized that incorporating adaptive excitation into high-speed 2PM could achieve deep, simultaneous voltage imaging of multiple neurons in vivo while remaining below the tissue heating threshold.

 

Methods: The authors developed a dual-plane adaptive excitation two-photon microscope combining a polygon-galvanometer scanner with Pockels cell–based illumination control synchronized via an acquisition system and ScanImage. Data were acquired through ScanImage and processed with MATLAB, while system synchronization and digitization were coordinated through a vDAQ. Neurons expressing the genetically encoded voltage indicator ASAP5 were imaged in the visual cortex of awake mice.

 

Results: Adaptive excitation enhanced signal collection 40–50-fold compared to conventional imaging at identical power, enabling detection of supra- and subthreshold neuronal activities at depths up to 635 µm with signal-to-noise ratios above 9. Dual-plane imaging across 80–115 µm axial separations showed minimal crosstalk and synchronized subthreshold oscillations in deep layers.

 

Conclusions: Adaptive excitation permits noninvasive, high-speed, deep two-photon voltage imaging of multiple neurons, approaching the theoretical performance limit for this technique and offering straightforward implementation on standard microscopes.

Mok AT, Wang T, Zhao S, Kolkman KE, Wu D, Ouzounov DG, Seo C, Wu C, Fetcho JR, Xu C. A large field-of-view, single-cell-resolution two- and three-photon microscope for deep and wide imaging. eLight 2024;4:20. doi: 10.1186/s43593-024-00076-4.

 

Background: Large field-of-view (LFOV) deep imaging at single-cell resolution is essential for understanding brain-wide neuronal activity, yet traditional two-photon microscopy (2PM) is limited to superficial cortical layers, and three-photon microscopy (3PM) typically suffers from restricted imaging speed and area. To address these limitations, the authors designed an integrated system capable of deep, wide and high-resolution imaging across multiple cortical and subcortical regions.

 

Hypothesis: This study hypothesized that a combination of adaptive excitation, beamlet scanning and polygon-based high-speed scanning could enable simultaneous two- and three-photon imaging with large field-of-view and single-cell resolution at depths previously inaccessible to conventional systems.

 

Methods: The authors developed a custom multiphoton microscope (“DEEPscope”) that integrates adaptive excitation modules, a beamlet generation delay line and polygon-galvo scanners. The setup incorporated a vDAQ and used ScanImage for synchronized signal acquisition and virtual channel processing. Motion correction and neuron segmentation were performed with Suite2p, while three-dimensional reconstructions were generated in Imaris. Animal imaging experiments were conducted in awake transgenic mice and anesthetized zebrafish.

 

Results: The system achieved a 3.23-mm field of view with single-cell resolution, enabling imaging of cortical layer 6 and hippocampal neurons through intact cortex. It recorded activity from 917 neurons at 600 µm depth and from over 4,500 neurons during dual two- and three-photon imaging. Whole-brain zebrafish imaging resolved cellular nuclei to depths exceeding 1,090 µm.

 

Conclusions: This platform demonstrates a powerful, scalable approach for large-scale, deep and high-speed multiphoton imaging, advancing system-level investigations of neural circuitry.