Optica

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.

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial sampling, respectively. In this Letter, we introduce a novel dual-axis scan unit for OPM that enables rapid and high-resolution volumetric imaging throughout a volume of 800 x 500 x 200 microns. This enables imaging of model organisms, such as zebrafish embryos, with subcellular resolution. Furthermore, we combined this microscope with a real-time and multi-perspective projection imaging technique to increase the volumetric interrogation rate to more than 10 Hz.

Head-mounted miniaturized two-photon microscopes are powerful tools to record neural activity with cellular resolution deep in the mouse brain during unrestrained, free-moving behavior. Two-photon microscopy, however, is traditionally limited in imaging frame rate due to the necessity of raster scanning the laser excitation spot over a large field-of-view (FOV). Here, we present two multiplexed miniature two-photon microscopes (M-MINI2Ps) to increase the imaging frame rate while preserving the spatial resolution. Two different FOVs are imaged simultaneously and then demixed temporally or computationally. We demonstrate large-scale (500x500 m2 FOV) multiplane calcium imaging in visual cortex and prefrontal cortex in freely moving mice for spontaneous activity and auditory stimulus evoked responses. Furthermore, the increased speed of M-MINI2Ps also enables two-photon voltage imaging at 400 Hz over a 380x150 m2 FOV in freely moving mice. M-MINI2Ps have compact footprints and are compatible with the open-source MINI2P. M-MINI2Ps, together with their design principles, allow the capture of faster physiological dynamics and population recordings over a greater volume than currently possible in freely moving mice, and will be a powerful tool in systems neuroscience.

Coherent Raman scattering imaging has provided inherent chemical information of biomolecules without the need for any external labels.1-3 However, its working depth in deep-tissue imaging is extremely shallow because both the intrinsic scattering cross-section and image contrast are so small that even weak perturbation of the pump and Stokes beam focusing by the complex tissue causes the loss of the resolving power.4,5 Here, we propose a deep-tissue coherent Raman scattering (CRS) microscopy equipped with an advanced adaptive optics (AO) system measuring complex tissue aberration from elastic backscattering. Using this label-free AO-CRS microscopy, we demonstrate the vibrational imaging of lipid-rich substances such as myelin inside the mouse brain even through the thick and opaque cranial bones.

Light-sheet imaging of 3D objects with high spatial resolution remains an open challenge because of the trade-off between field-of-view (FOV) and axial resolution originating from the diffraction of light. We developed acoustic light-sheet microscopy (acoustic LSM), which actively manipulates the light propagation inside a large sample to obtain wide-field microscopic images deep inside a target. By accurately coupling a light-sheet illumination pulse into a planar acoustic pulse, the light-sheet can be continuously guided over large distances. We imaged a fluorescence-labeled transparent mouse brain for the FOVs of 19.3 x 12.4 mm2 and 9.7 x 5.9 mm2 with resolved microstructures and single cells deep inside the brain. Acoustic LSM creates new opportunities for the application of light-sheet in the field of industry to basic science. One Sentence SummaryAn acoustic-optical method overcomes a trade-off between field-of-view and axial resolution in light-sheet microscopy.

We demonstrate a multi-focal multi-photon volumetric microscopy via combination of 32-beam parallel lateral-scanning, a 70-kHz axial-scanning acoustic lens, and a 32-channel photodetector, enabling unprecedented data rate (2-10 GHz) and >500-volumes/second imaging speed over ~200x200x200-m3.

Many fundamental biological processes--spanning immune-tumor interactions, neuronal signaling, and microvascular flow--exhibit fast, multiscale dynamics among diverse cell types within three-dimensional tissue environments. Capturing such activity requires imaging systems that simultaneously achieve high temporal resolution, multicolor capability, and volumetric coverage over large fields of view (FOVs). However, existing modalities remain limited by trade-offs among imaging speed, spectral capacity, depth of field (DOF), and spatial resolution. Here, we present Dual-Channel Event Microscopy (DEM), which integrates digital micromirror device (DMD)-based pulsed illumination, extended-DOF optics, and event-based sensing for ultrafast, dual-channel volumetric imaging across a 2.3 x 1.3 mm2 FOV with an effective 200 {micro}m DOF. Using dual-color fluorescent phantoms and microsphere flow assays, DEM achieves accurate spectral separation and reconstruction of rapid motion at kilohertz frame rates. In vivo, DEM enables simultaneous visualization of neutrophils and premalignant tumors in freely swimming zebrafish. In immobilized specimens, it provides robust, sensor-level optical sectioning near the heart, suppressing diffuse background to reveal fine vascular networks and active blood circulation into and out of the cardiac chambers. DEM further enables quantitative mapping of blood-flow dynamics in the zebrafish tail, resolving arterial-venous differences and capturing heartbeat-driven oscillations that reflect cardiac pumping with high temporal fidelity. By uniting ultrafast acquisition, dual-channel capability, volumetric coverage, and intrinsic optical sectioning within a single event-driven architecture, DEM offers a powerful platform for visualizing rapid multicellular interactions and physiological dynamics in living systems.

3D tracking and localization of particles, typically fluorescently labeled biomolecules, provides a direct means of monitoring cellular transport and communication. However, sample-induced wavefront distortions of emitted fluorescent light as it passes through the sample and onto the detector often yield point spread function (PSF) aberrations, presenting an important challenge to 3D particle tracking using pre-calibrated PSFs. PSF calibration is typically performed outside cellular samples, ignoring sample-induced aberrations, which can result in localization errors on the order of tens to hundreds of nanometers, ultimately compromising sub-diffraction limited tracking. In practice, correcting sample-induced aberrations currently requires sample-specific hardware adjustments, such as adaptive optics. Yet, information on sample-induced aberrations and PSF shape can be directly decoded from data collected using a 3D imaging setup. To this end, we propose a framework for simultaneous particle tracking, pupil function learning, and PSF reconstruction directly from the input data themselves. To accomplish this, we operate within a Bayesian paradigm, placing continuous 2D priors on all possible pupil phase and amplitudes warranted by the data without limiting ourselves to a finite Zernike set-thereby allowing capture of intricate pupil phase details. We benchmark our framework using a wide range of synthetic and experimental data from static to diffusing particles, and generalize to multiple diffusing particles with overlapping PSFs. Further, as a result of simultaneous particle tracking, phase retrieval, and PSF reconstruction, we retrieve the pupil phase with errors smaller than 10% under a range of realistic scenarios, while restoring sub-diffraction limited localization precisions of 10-25 nm and 20-50 nm in lateral and axial directions, respectively.

AO_SCPLOWBSTRACTC_SCPLOWSuper-resolution fluorescence microscopy has transformed the study of biological structures and functions beyond the diffraction limit. Unlike fluorescence methods, label-free chemical imaging, mostly based on Raman and infrared spectroscopy, provides intrinsic molecular contrast, enabling the study of biomolecules, nanostructures, drug molecules, and metabolites that cannot be easily tagged. However, while a wide range of fluorescence-based super-resolution techniques are well-established, extending super-resolution to label-free chemical imaging has remained challenging due to low signal levels and limited resolution improvement. Super-resolution stimulated Raman scattering microscopy (SRS) is most promising due to its high sensitivity and imaging speed. Similar to fluorescence, existing SRS super-resolution approaches are mostly based on either photoswitching/saturation of molecular labels or sample expansion, which suffers from poor sensitivity due to limitations in labeling density or signal dilution, respectively. Moreover, axial resolution is typically much worse than lateral resolution, yet most super-resolution SRS techniques focused on improving lateral resolution. In this work, we combine stimulated Raman scattering (SRS) with 4Pi-interferometry to significantly improve the axial resolution by nearly 7-fold. We report on the characterization of improvements in imaging sensitivity and axial resolution using 80 nm polystyrene beads. Harnessing the improved axial resolution, we demonstrate super-resolution 4Pi-SRS imaging in resolving small lipid droplet structures in mammalian cells and lipid membranes in E. coli cells. Because 4Pi-SRS uses interferometry to improve axial resolution, it is completely orthogonal to all previous super-resolution SRS techniques, including visible excitation, photoswitching, sample expansion, and computational approaches, thus it is straightforward to combine them to achieve much higher resolution chemical imaging than currently possible.

Two-photon light sheet microscopy offers great potential for a range of biological applications. Still, its practical implementation is impeded by the high cost of laser sources, the complexity of construction, and the challenges associated with adapting to existing microscope setups. Here, we release an open-source design that addresses these limitations by providing detailed building instructions for transforming a brightfield microscope into a versatile one- and two-photon light sheet system. Our design incorporates a specially designed broadband hollow core fiber, enabling the simultaneous utilization of visible laser alongside an expansive pulsed laser source from another setup. This integration allows for uncompromised image resolution and speed. Furthermore, the design reduces the complexity of construction, alignment, and overall cost, thereby significantly enhancing the accessibility of this technology (https://github.com/LJPZebra/OLU).

Three-dimensional (3D) volumetric imaging of the human retina is instrumental to monitor and diagnose blinding conditions. Although coherent retinal imaging is well established by optical coherence tomography, it is still a large void for incoherent volumetric imaging in the human retina. Here, we report confocal oblique scanning laser ophthalmoscopy (CoSLO), to fill that void and harness incoherent optical contrast in 3D. CoSLO uses oblique scanning laser and remote focusing to acquire depth signal in parallel, avoid the lengthy z-stacking, and image a large field of view (FOV). In addition, confocal gating is introduced by a linear sensor array to improve the contrast and resolution. For the first time, we achieved incoherent 3D human retinal imaging with >20{degrees} viewing angle within only 5 seconds. The depth resolution is [~]45 microns in vivo. We demonstrated label-free incoherent contrast by CoSLO, revealing unique features in the retina. CoSLO will be an important technique for clinical care of retinal conditions and fundamental vision science, by offering unique volumetric incoherent contrasts.

Expansion microscopy, a super-resolution fluorescence microscopy technique in which samples are expanded up to [~]8,000 times (after 20-fold expansion) their original volume, places high demands on the microscopes used to image the expanded samples. To reveal nanoscale cellular ultrastructure in meaningful sample volumes, the instruments need to feature a large field of view and working distance. Simultaneously, they need to offer a high three-dimensional resolution to avoid counteracting the resolution improvement achieved by the expansion process. Here, we present pan-ASLM, a high resolution, large field-of-view light-sheet microscope developed for expanded samples, based on the Axially Swept Light Sheet Microscopy (ASLM) technique. pan-ASLM allows imaging over a 640 {micro}m x 640 {micro}m field of view with lateral and axial resolutions of 566 nm and 457 nm, respectively, and features an image acquisition speed of up to 20 fps (183 Mvoxels/sec). It offers [~]1200x higher imaging speed, a [~]7x larger field of view, and [~]2x better axial resolution than the standard confocal microscopes typically used for expanded samples. We validate the new microscope design through imaging of pan-expanded HeLa cells as well as mouse kidney and brain tissue.

In vivo imaging of dynamic sub-cellular brain structures in Drosophila melanogaster is key to understanding several phenomena in neuroscience. However, its implementation has been hindered by a trade-off between spatial resolution, speed, photobleaching, phototoxicity, and setup complexity required to access the specific target regions of the small brain of Drosophila. Here, we present a single objective light-sheet microscope, customized for in vivo imaging of adult flies and optimized for maximum resolution. With it, we imaged the axonal projections of small lateral ventral neurons (known as s-LNvs) in intact adult flies. We imaged the plasma membrane, mitochondria, and dense-core vesicles with high spatial resolution up to 370 nm, ten times lower photobleaching than confocal microscopy, lower invasiveness and complexity in sample mounting than alternative light-sheet technologies, and without relying on phototoxic pulsed infrared lasers. This unique set of features paves the way for new long-term, dynamic studies in the brains of living flies.

We have demonstrated 30-Hz three-photon imaging using a single 24-MHz mode-locked Cr:forsterite oscillator with a center wavelength at 1260 nm. By managing the dispersion distribution in the resonator using double-chirped mirrors, we have produced 32-fs pulses with 22-nJ pulse energy. Using the oscillator as a driving source, we have realized multi-color three-photon images using a GFP-labeled Drosophila brain and an AF647-labeled mouse brain. To demonstrate the capability of deep-tissue imaging, we have obtained a 10-times higher SBR from the three-photon images than the two-photon results at different depths in a GFP-labeled Drosophila brain dissection. Furthermore, we have shown the impact of excitation pulse width on three-photon deep-tissue imaging. Our results indicate the superiority of using shorter pulses for deeper-tissue imaging, especially in the Drosophila brain. In addition, we have recorded the three-photon calcium imaging in vivo from the Drosophila mushroom body in response to external electric shocks. We believe our demonstration provides a robust approach for high-speed three-photon microscopy applications, especially for intravital investigations in the Drosophila brain.

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Although several open-source, easy-to-assemble light-sheet microscope platforms already exist--such as mesoSPIM, OpenSPIM, and OpenSpin--they are optimized for imaging large specimens and lack the resolution required to visualize sub-cellular features, such as organelles or cytoskeletal architectures. In contrast, Latice Light-Sheet Microscopy (LLSM) achieves the resolution necessary to resolve such fine structures but, in its open-source implementation, can be alignment- and maintenance-intensive, often requiring specialist expertise. To address this gap, we developed Altair-LSFM, a high-resolution, open-source, sample-scanning light-sheet microscope specifically designed for sub-cellular imaging. By optimizing the optical pathway in silico, we created a custom baseplate that greatly simplifies alignment and assembly. The system integrates streamlined optoelectronics and optomechanics with seamless operation through our open-source software, navigate. Altair-LSFM achieves lateral and axial resolutions of approximately 235 nm and 350 nm, respectively, across a 266-micron field of view after deconvolution. We validate the systems capabilities by imaging sub-diffraction fluorescent nanospheres and visualizing fine structural details in mammalian cells, including microtubules, actin filaments, nuclei, and Golgi apparatus. We further demonstrate its live-cell imaging capabilities by visualizing microtubules and vimentin intermediate filaments in actively migrating cells.

Raman microscopy provides chemically selective imaging by exploiting intrinsic vibrational properties of specimens. Yet, a fast acquisition, low phototoxicity, and non-specific (to a vibrational/electronic mode) super-resolution method has been elusive for tissue imaging. We demonstrate a single-pixel-based approach, combined with robust structured illumination, that enables fast super-resolution in stimulated Raman scattering microscopy at low power levels. The methodology is straightforward to implement and compatible with thick biological specimens, therefore paving the way for probing complex biological systems when exogenous labelling is challenging.

Commercial Ti:Sapphire femtosecond lasers used for conventional two-photon microscopy typically operate at a [~]80 MHz repetition rate. However, this frequency is often suboptimal for cortical tissue imaging, where lower rates of 20-40 MHz are considered ideal. However, achieving these lower frequencies has remained a significant technical and financial challenge. Here, we present a compact, cost-effective resonant electro-optic modulator that halves the laser repetition rate to 40 MHz. When imaging neurons in the macaque visual cortex in vivo, this 40 MHz configuration yielded a >2-fold increase in fluorescence intensity and a [~]3 dB improvement in signal-to-noise ratio (SNR) for both green (GCaMP5G) and red (mScarlet) indicators. This enhancement proved particularly pronounced in deep-tissue imaging. Furthermore, the system demonstrated excellent long-term stability and induced no detectable phototoxicity. This simple and robust device represents a powerful upgrade for conventional two-photon microscopes, significantly enhancing imaging quality for the investigation of neural circuits within scattering brain tissue.

Reliable and reproducible measurement of spatial resolution is essential for validating and comparing the performance of two-photon microscopy systems. We present fluorescent nanodiamonds as robust, photostable, reusable, and biocompatible probes for benchmarking spatial resolution. Owing to their nanoscale dimensions and stable fluorescence, nanodiamonds act as near-ideal point-like emitters, enabling accurate characterisation of the point spread function across varying imaging conditions. We demonstrate that nanodiamond-based phantoms serve as a reliable alternative to conventional fluorescent beads embedded in agarose, while at the same time offering advantages in stability and optical properties. Our results position nanodiamond phantoms as a next-generation calibration material that bridges ease of use and reproducibility, advancing quantitative imaging and cross-platform comparability in modern fluorescence microscopy.

Microscopes generally exhibit superior performance in 2D compared to 3D. Fluorescence super-resolution imaging often intensifies this discrepancy, such as with the gold-standard vortex-based stimulated emission depletion (STED) microscope, which narrows the point-spread function only laterally. In this study, we developed a semi-analytical theory based on the Nijboer-Zernike expansion, conducted simulations and performed experiments to establish the merits of the alternative bivortex-based STED. We find that this mode reduces the axial-lateral resolution mismatch and effectively emulates noisier multi-beam approaches by providing access to point-spread function geometries that are strictly forbidden to the two conventional single-beam modes, 2D-STED and z-STED. Notably, theory and experiment indicate that, besides filling the gap, bivortex STED delivers a higher signal-to-background than the two modes it bridges.