Optica

In vivo imaging of large-scale neuron activity plays a pivotal role in unraveling the function of the brains network. Multiphoton microscopy, a powerful tool for deep-tissue imaging, has received sustained interest in advancing its speed, field of view and imaging depth. However, to avoid thermal damage in scattering biological tissue, field of view decreases exponentially as imaging depth increases. We present a suite of innovations to overcome constraints on the field of view in three-photon microscopy and to perform deep imaging that is inaccessible to two-photon microscopy. These innovations enable us to image neuronal activities in a [~]3.5-mm diameter field-of-view at 4 Hz with single-cell resolution and in the deepest cortical layer of mouse brains. We further demonstrate simultaneous large field-of-view two-photon and three-photon imaging, subcortical imaging in the mouse brain, and whole-brain imaging in adult zebrafish. The demonstrated techniques can be integrated into any multiphoton microscope for large-field-of-view and system-level neural circuit research.

ABSTACTIn neuroscience, diffraction limited two-photon (2P) microscopy is a cornerstone technique that permits minimally invasive optical monitoring of neuronal activity. However, most conventional 2P microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing divergent beam optics (DBO), we present an ultra-low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view that also maintains single-cell resolution. We show that this implementation increases both the space-bandwidth product and effective excitation power, and allows for straight-forward tailoring of the point-spread-function. Moreover, rapid laser-focus control via an electrically tunable lens now allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, and fully compatible with a wide range of existing 2P customizations, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in mice, zebrafish and fruit flies.

Ultrafast two-photon fluorescence microscopy (2PFM) based on free-space angular-chirp-enhanced delay (FACED) enables megahertz line scanning and kilohertz frame rates for in vivo brain imaging. However, optical aberrations from the imaging system and brain tissue degrade spatial resolution, signal, and contrast at depth. Here we integrate adaptive optics (AO) with FACED 2PFM to achieve synapse-resolving ultrafast imaging in the living mouse brain. Because FACED generates a one-dimensional array of temporally delayed, spatially separated excitation foci at 1 gigahertz, we developed a focus-averaging, frequency-multiplexed aberration measurement method that simultaneously measures and corrects the average aberration across all FACED foci using a segmented deformable mirror. We validated the accuracy of our method in correcting both system and artificial aberrations. When applied to in vivo morphological imaging of the mouse brain, AO enhances resolution, signal, contrast of dendritic shafts, spines, and boutons. Functionally, AO improves cerebral blood flow imaging by increasing plasma signal and kymograph contrast over large fields of view; when used for glutamate imaging, it amplifies transient amplitudes and reveals visually evoked glutamate release that were undetectable without correction. Together, these results establish AO-FACED 2PFM as a powerful approach that combines ultrafast imaging with high spatial resolution for structural and functional imaging in the living mouse brain.

Three-photon (3P) microscopy enables functional non-invasive single-cell resolution imaging at greater depths than any other technique. A key challenge of deep imaging is tissue-induced optical aberration, which reduces the excitation confinement. Adaptive optics use deformable mirrors to compensate for optical distortions, hence correcting these aberrations. Here, we present a practical adaptive optics-assisted 3P imaging system for functional imaging in the mouse brain during behavior. We introduce hierarchical corrections that sequentially target aberrations caused by the microscope system, the cranial window, and tissue depth. We demonstrate the utility of this strategy in the prelimbic cortex, where large vasculature near the midline causes aberrations, and in the lateral somatosensory cortex, where side access leads to distinct wavefront distortions. In both regions, adaptive optics significantly improved imaging performance, restoring cellular visibility near vasculature and enhancing signal-to-noise ratio. Our work provides a practical framework for utilizing adaptive optics to improve 3P imaging during behavior.

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 m into the mouse spinal cord, in vivo. We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 m of the murine spinal cord, in vivo.

Switching laser mode (SLAM) microscopy is a promising method for achieving super resolution while maintaining compatibility with two photon imaging at depth and in vivo. SLAM microscopes typically employ multiple paths for generating the requisite spot-like and donut-like beams; however, having two paths necessitates sub-wavelength-scale alignment which is prone to differential drift, causing degradation of the image quality. Here we demonstrate a single aperture, inline SLAM microscope which makes use of one phase element and polarization switching to generate colinear radially polarized and azimuthally polarized vector beams, which focus to a spot and a donut, respectively. By tailoring the spatial profile of the electric field at the back aperture of the microscope objective, we ensure that the resolution of the spot-like beam is comparable to conventional Gaussian beam imaging. Through subtraction of the two images, we demonstrate a 1.5x narrower focal spot and a resolution of [~]0.28{lambda} corresponding to [~]290 nm. Accordingly, this method is of great utility for imaging with sub-diffraction-limited resolution at depth in living tissue.

Recording activity from large cell populations in deep neural circuits is essential for understanding brain function. Three-photon (3P) imaging is an emerging technology that allows for imaging of structure and function in subcortical brain structures. However, increased tissue heating, as well as the low repetition rate sources inherent to 3P imaging, have limited the fields of view (FOV) to areas of [≤] 0.3 mm2. Here we present a Large Imaging Field of view Three-photon (LIFT) microscope with a FOV of >3 mm2. LIFT combines high numerical aperture (NA) optimized sampling, using a custom scanning module, with deep learning-based denoising, to enable population imaging in deep brain regions. We demonstrate non-invasive calcium imaging in the mouse brain from >1500 cells across CA1, the surrounding white matter, and adjacent deep layers of the cortex, and show population imaging with high signal-to-noise in the rat cortex at a depth of 1.2 mm. The LIFT microscope was built with all off-the-shelf components and allows for a flexible choice of imaging scale and rate.

Brain research is an area of research characterized by its cutting-edge nature, with brain mapping constituting a crucial aspect of this field. As sequencing tools have played a crucial role in gene sequencing, brain mapping largely depends on automated, high-throughput and high-resolution imaging techniques. Over the years, the demand for high-throughput imaging has scaled exponentially with the rapid development of microscopic brain mapping. In this paper, we introduce the novel concept of confocal Airy beam into oblique light-sheet tomography named CAB-OLST. We demonstrate that this technique enables the high throughput of brain-wide imaging of long-distance axon projection for the entire mouse brain at a resolution of 0.26 m x 0.26 m x 1.06 m in 58 hours. This technique represents an innovative contribution to the field of brain research by setting a new standard for high-throughput imaging techniques.

Advances in genetically encoded fluorescent indicators have enabled increasingly sensitive optical recordings of neural activity. However, light scattering in the mammalian brain tissue restricts optical access to deeper regions. To address this limitation, researchers often employ implanted gradient-index (GRIN) lenses to reach deep brain areas. Nevertheless, the severe optical aberrations of GRIN lenses significantly reduce the effective field of view (FOV). In this work, we present a simple and robust imaging approach that combines low-NA telecentric scanning (LNTS) of laser excitation with high-NA fluorescence collection to increase the FOV. This configuration effectively eliminates common aberrations such as astigmatism and field curvature, resulting in a FOV [~]100% as large as the GRIN lens facet area -- corresponding to a [~]400% increase in imaging area compared with conventional approaches. We validate this method through both structural and functional in vivo imaging. The highly consistent imaging performance, fully maximized imaging FOV, and the very simple optical design make this method well-suited for broad dissemination in neuroscience research.

Imaging neural structures deep in brain tissue is central to understanding brain function, yet remains fundamentally limited by strong optical scattering and the requirement for accurate three-dimensional (3D) optical sectioning. Laser-scanning microscopy is a promising technique for brain imaging; however, maintaining excitation focus integrity in scattering media while preserving axial confinement poses a persistent photonic challenge. Here we introduce the optical pin, an ultrashort excitation regime engineered at the angular-spectrum level to address this limitation. By broadening the transverse angular bandwidth of a Bessel-type field while preserving its conical momentum-space architecture, the optical pin introduces a controlled longitudinal wave-vector spread that compresses the axial interference length to the micrometer scale, restoring Gaussian-like sectioning without sacrificing multi-angle interference. This excitation design yields substantially enhanced imaging performance, including [~]1.5-fold contrast improvement and [~]2.6-fold increased robustness to scattering. We validate the approach across transparent, scattering, and biological specimens, including bead phantoms, C. elegans, and mouse brain tissue. As a system-level excitation strategy, the optical pin is readily compatible with existing laser-scanning microscopy platforms and is particularly suited for scattering-limited brain imaging.

Adaptive optics (AO) restore ideal imaging performance in complex samples by measuring and correcting optical aberrations, but often require custom-built microscopes with carefully aligned wavefront sensing/shaping devices and can be susceptible to sample motion. Here we describe NeAT, a computational framework using neural fields for AO two-photon fluorescence microscopy. NeAT estimates wavefront aberration and recovers sample structure from a 3D image stack without requiring external datasets for training. Incorporating motion correction in learning and correcting conjugation errors commonly found in commercial microscopes, NeAT is designed for deployment in biological laboratories for in vivo imaging. We validate NeATs performance using a custom-built microscope with a wavefront sensor under varying signal-to-noise ratios, aberration, and motion conditions. With a commercial microscope, we demonstrate real-time aberration correction for in vivo morphological and functional imaging in the living mouse brain, with NeAT improving signal and accuracy of glutamate and calcium imaging of synapses and neurons.

High-resolution imaging under physiological conditions is essential for studying biological mechanisms and disease processes. However, achieving this goal remains challenging due to optical aberrations and scattering from heterogeneous tissue structures, compounded by motion artifacts from awake animals. In this study, we developed a rapid and accurate adaptive optics system called multiplexing digital focus sensing and shaping (MD-FSS) for deep-tissue multiphoton microscopy. Under two-photon excitation, MD-FSS precisely measures the aberrated point spread function in approximately 0.1 s per measurement, effectively compensating for both aberrations and scattering to achieve subcellular resolution in deep tissue. Using MD-FSS integrated with two-photon microscopy, we achieved high-resolution brain imaging through thinned or optically cleared skull windows, two near noninvasive methods to access mouse brain, reaching depths up to 600 m below the pia in awake behaving mice. Our findings revealed significant differences in microglial functional states and microvascular circulation dynamics between awake and anesthetized conditions, highlighting the importance of studying brain function in awake mice through noninvasive methods. We captured functional imaging of fine neuronal structures at subcellular level in both somatosensory and visual cortices. Additionally, we demonstrated high-resolution imaging of microvascular structures and neurovascular coupling across multiple cortical regions and depths in the awake brain. Our work shows that MD-FSS robustly corrects tissue-induced aberrations and scattering through rapid PSF measurements, enabling near-noninvasive, high-resolution imaging in awake, behaving mice.

Mask-based lensless fluorescence microscopy is a compact, portable imaging technique promising for biomedical research. It forms images through a thin optical mask near the camera without bulky optics, enabling snapshot three-dimensional imaging and a scalable field of view (FOV) without increasing device thickness. Lensless microscopy relies on computational algorithms to solve the inverse problem of object reconstruction. However, there has been a lack of efficient reconstruction algorithms for large-scale data. Furthermore, the entire FOV is typically reconstructed as a whole, which demands substantial computational resources and limits the scalability of the FOV. Here, we developed DeepLeMiN, a lensless microscope with a custom designed optical mask and a multi-stage physics-informed deep learning model. This not only enables the reconstruction of localized FOVs, but also significantly reduces the computational resource demands and facilitates real-time reconstruction. Our deep learning algorithm can reconstruct object volumes over 4x6x0.6 mm3, achieving lateral and axial resolution of [~]10 {micro}m and [~]50 {micro}m respectively. We demonstrated significant improvement in both reconstruction quality and speed compared to traditional methods, across various fluorescent samples with dense structures. Notably, we achieved high-quality reconstruction of 3D motion of hydra and the neuronal activity with cellular resolution in awake mouse cortex. DeepLeMiN holds great promise for scalable, large FOV, real-time, 3D imaging applications with compact device footprint.

Intravital multi-photon imaging of the bone marrow is crucial to the study of cellular dynamics, communication with the microenvironment and functions, however, imaging of deep tissue areas is challenging and minimally invasive methods for deep-marrow imaging in intact long bones are needed. We developed a high pulse energy 1650 nm laser prototype, which permits to surpass >100 {micro}m thick cortical bone and to perform three-photon microscopy (3PM) in more than 400 {micro}m depth in the marrow cavity of intact mouse tibia in vivo. Its unique 3 and 4 MHz laser repetition rates allowed us to analyze motility patterns of rare cells over large fields of view deep within the unperturbed marrow. In this way, we found a bi-modal migratory behavior of marrow plasma cells. Besides, the analysis of third harmonics generation (THG) in the tibia identified this signal to be a label-free indicator of the abundance of cellular organelles, in particular the endoplasmic reticulum, reflecting protein biosynthesis capacity. We found that only one third of the plasma cells in the tibia marrow of adult mice have a strong THG signal and, thus, a high protein synthesis capacity, while the other two thirds of plasma cells display a low THG signal. Finally, we identified an inverse link between migratory behavior and THG signal strength in marrow plasma cells. As in these cells, the protein biosynthesis capacity indicated by a strong THG signal is mainly associated with antibody secretion, we could relate motility to functional states of plasma cells in vivo. Our 3PM method retains the ability to connect cellular dynamics to protein biosynthesis capacity in various marrow cell types beyond plasma cells, as THG is a ubiquitous signal, opening new perspectives on understanding how tissue microenvironment impacts on cellular functions in the bone marrow.

All-optical interrogation, based on high-resolution two-photon stimulation and imaging, has emerged as a potentially transformative approach in neuroscience, allowing for the simultaneous precise manipulation and monitoring of neuronal activity across various model organisms. However, the unintended excitation of light-gated ion channels such as channelrhodopsin (ChR) during two-photon calcium imaging with genetically encoded calcium indicators (GECIs) introduces artifactual neuronal perturbation and contaminates neural activity measurements. In this study, we propose an active pixel power control (APPC) approach, which dynamically adjusts the imaging laser power at each scanning pixel, to address the challenge. We aim to achieve simultaneous two-photon optogenetic manipulation and calcium imaging with a single femtosecond laser, while minimizing the crosstalk between manipulation and imaging. To study this technologys capabilities, we applied it to the larval zebrafish brain in vivo. Our results demonstrate that the APPC approach preserves GECI signal quality while suppressing optogenetic artifacts significantly. This enhances the accuracy of neural circuit dissection and advances the precision of all-optical interrogation, offering a robust framework for probing neural circuit dynamics and causality in vivo with high fidelity, potentially across various model organisms. Importantly, this technology can be seamlessly integrated with commonly used two-photon microscope systems in laboratories worldwide.

Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

Observing the activity patterns of large neural populations throughout the brain is essential for understanding brain function. However, capturing neural interactions across widely distributed brain regions from both superficial and deep cortical layers remains challenging with existing microscopy technologies. Here, we introduce a state-of-the-art two-photon microscopy system, ULTRA, capable of single-cell resolution imaging across an ultra-large field of view (FOV) exceeding 50 mm{superscript 2}, enabling deep and very wide field in vivo imaging. To demonstrate its capabilities, we conducted a series of experiments under multiple imaging conditions, successfully visualizing brain structures and neuronal activities spanning a spatial range of over 7 mm from superficial layers to depths of up to 900 m, while covering a volume of 45.24 mm3 in the mouse brain. This versatile imaging platform overcomes traditional spatial constraints, providing a powerful tool for comprehensive exploration of neuronal circuitry over extensive spatial scales with cellular resolution.

Disruptions in capillary flow have the potential to drive pathology across numerous diseases. But our understanding of the temporal and spatial dynamics of these events are hindered by slow volumetric imaging rates and the reliance on laborious manual analysis to process data. To address the challenges of increasing volumetric imaging speed, we use a custom-built Bessel beam two-photon microscope for efficient volumetric imaging of the capillary network. We demonstrate its ability to continuously monitor roughly 200 capillaries for capillary flow stoppages (i.e. stalling events) at a frame rate of approximately 0.5 Hz and develop a semi-automated correlation-based approach for identifying these stalling events. We applied our system and algorithm in a photothrombotic model of stroke and show elevated levels of stalling 1-week post-stroke in regions both within and outside of the stroke region, demonstrating that stalling may have impacts on stroke recovery that extend past the acute stage.

Widefield fluorescence microscopy is widely used for imaging at subcellular resolution, but its performance in complex samples is degraded by optical aberrations. Because aberrations can vary spatially across the field of view (FOV), accurate aberration measurement and correction at multiple FOV locations are essential for achieving high-quality imaging over large areas. Here, we introduce parallel frequency-multiplexed aberration measurement (PFAM) to perform massively parallel aberration measurements across an extended FOV. We validated PFAM using fluorescent beads and demonstrated simultaneous measurement and effective correction of spatially varying aberrations at 125 FOV locations. To address the challenges of wavefront sensing in complex samples, we further developed PFAM-SIFT by integrating structured illumination, thereby achieving robust aberration measurement in both brain slices and the mouse brain in vivo. Together, PFAM and PFAM-SIFT provide accurate and scalable wavefront sensing solutions for widefield imaging, enabling simultaneous aberration measurement of spatially varying aberrations in complex biological samples.

Two-photon microscopy, combined with appropriate optical labeling, has enabled the study of structure and function throughout nervous systems. This methodology enables, for example, the measurement and tracking of sub-micrometer structures within brain cells, the spatio-temporal mapping of spikes in individual neurons, and the spatio-temporal mapping of transmitter release in individual synapses. Yet the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300 to 400 {micro}m of neocortex. To obviate this limitation, we measure the wavefront at the focus of the excitation beam and utilize adaptive optics that alters the incident wavefront to achieve an improved focal volume. We describe the constructions, calibration, and operation of a two-photon microscopy that incorporates adaptive optics to restore diffraction-limited resolution throughout the nearly 900 {micro}m depth of mouse cortex. Our realization utilizes a guide star formed by excitation of red-shifted dye within the blood serum to directly measure the wavefront. We incorporate predominantly commercial optical, optomechanical, mechanical, and electronic components; computer aided design models of the exceptional custom components are supplied. The design is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in mouse neocortex by imaging the morphology of somatostatin-expressing neurons at 700 {micro}m beneath the pia, calcium dynamics of layer 5b projection neurons, and glutamate transmission to L4 neurons.