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.

We present cleared-tissue direct-view oblique plane microscopy (CtDvOPM), which enables optically sectioned subcellular resolution imaging of centimetre-scale tissues at high-throughput over the full range of clearing media refractive indices (n = 1.33-1.56). CtDvOPM can image conventionally-mounted expanded, aqueous or non-aqueous cleared tissue samples at up to 2 {micro}m lateral by 14 {micro}m axial resolution over a 10 mm x 10 mm x 25 mm sample volume without image tiling, at up to 400 million voxels per second.

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.

Light-sheet microscopy (LSM) has revolutionized bioimaging by delivering high-contrast volumetric resolution with minimal photodamage. Spatial wavefront shaping, used to gen{-}erate lattice and Airy light-sheets, has been particularly effective in advancing LSM be{-}yond the Rayleigh limit. Despite its broad adoption, most LSM implementations rely on rigid dual-objective geometries that complicate sample handling and impose a trade-off between imaging field of view (FoV) and axial resolution. Here, we introduce space-time light-sheet microscopy (ST-LSM), a single-objective strategy that exploits space-time (ST) correlations for the first time. ST-LSM goes beyond separate spatial or temporal modulation to jointly modulate the spatiotemporal spectral structure of a pulse. This uniquely enabled light-sheets with wavelength-scale thickness over millimeter-scale dis{-}tances. When compared to state-of-the-art approaches, ST-LSM eliminates the dual-objective constraint, expands the sample-accessible volume by 25x, and increases the FoV by 10x without sacrificing sectioning resolution. We demonstrate the versatility of ST-LSM by using a single setup to image specimens across four orders of magnitude in size, from whole roots and developing embryos, down to mammalian cells with sub-cellular axial resolution. These results position ST-LSM as an accessible and high-performance optical microscopy platform at a variety of biological scales, by translating space-time wave-packet physics into a practical imaging modality.

Coherent structured illumination microscopy (c-SIM) is a synthetic aperture optical technique for sub-diffraction limit imaging that extends the utility of traditional SIM to non-fluorescent samples. Here, we present a complementary 5-beam implementation of c-SIM that provides enhanced optical sectioning compared to conventional quadrupolar illumination. Since our approach detects intensity images due to coherent light scattering, it avoids the complications associated with detecting complex fields. Through comparative measurements on calibration samples and live microalgae, we show that 5-beam c-SIM effectively suppresses coherent defocus effects, improving image quality while simultaneously providing a 2-fold lateral resolution improvement.

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.

Inverse-scattering methods enable label-free, quantitative visualization of a samples three-dimensional (3D) refractive index (RI), providing intrinsic and volumetric morphological contrast without exogenous labels. This is achieved by developing computational frameworks that reconstruct the samples 3D RI from a series of scattering measurements acquired under different data-capture conditions. Recent advances have demonstrated successful 3D RI reconstructions in multiple-scattering samples using angle-varying illuminations; however, these studies have primarily focused on non-absorptive samples. Here, we extend the multi-slice beam propagation (MSBP) inverse-scattering framework to reconstruct complex-valued RI, encompassing both the samples conventional RI (real part) and absorptivity (imaginary part). We show that reconstructing complex-valued RI makes the inverse problem ill-posed under angle-varying illumination alone, and that incorporating measurement diversity from both angle-varying illumination and sample defocus is necessary to ensure stable and accurate convergence. Experimental demonstrations were conducted on 1) dyed microsphere samples to characterize accuracy of reconstructed RI and absorptivity; and 2) diverse absorptive scattering samples to demonstrate biological utility. These results represent an important step for label-free volumetric imaging in biological tissue, which typically exhibits both scattering and absorption.

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.

The ability to record 3D neuronal activity with cellular resolution, high signal-to-noise ratio (SNR) and millisecond temporal resolution is a major challenge in neuroscience. One powerful method is random-access two-photon microscopy based on acousto-optic deflectors (AODs), which uses a holographically-shaped point spread function (PSF) scanned in 3D to maximize the sampling rate and SNR. However, this approach suffers from greater background contamination due to the holographically shaped PSF than standard two-photon microscopy with diffraction-limited PSF. To overcome this limitation, we implemented a new version of an AOD scanning system, which integrates temporal focusing. The complex spatiotemporal distortions encountered in this configuration, including a significant group delay dispersion associated with the pulse front tilt generated by the AOD, were compensated for by introducing an acousto-optic modulator before the AOD. We designed extended patterns by combining temporal focusing on one direction and holographic wavefront shaping in the perpendicular axis. Taking advantage of the AODs ability to shape the wavefront at the same speed as the scan, we were able to accurately superimpose the spatial and temporal foci over the entire field of view. Finally, we generated complex, extended two-photon excitation patterns by combining temporal focusing in one direction and holographic multiplexing in the perpendicular direction. These patterns provide significantly improved background rejection compared to 2D holographic patterns, thus offering promising prospects for in vivo recordings of neuronal activity in dense samples with improved SNR.

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).

Light-sheet fluorescence microscopy (LSFM) has revolutionized biological imaging by enabling high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360 degree omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This novel approach provides an advanced, scalable solution for large-volume imaging, facilitating detailed structural and functional studies across diverse biological applications.

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.

Structured Illumination Microscopy (SIM) provides imaging with spatial super-resolution, as well as optical sectioning capability, without relying on specialized fluorescent dyes. 2D and 3D variants of this method exist, but most bespoke implementations are 2D-SIM, because it is easier to realize and modify than 3D-SIM. 2D-SIM systems, however, often experience reconstruction artifacts, especially when pushing for high lateral spatial resolution in thicker samples. We present enhanced 2D-SIM, an approach to 2D-SIM where both, coarse patterns optimized for removing out-of-focus background, and fine patterns optimized for resolution improvement beyond the diffraction limit are used. In combination, this achieves 2D-SIM reconstructions with high contrast, spatial super-resolution, and significantly reduced reconstruction artifacts. We present the theoretical framework of this technique, and provide enhanced 2D-SIM imaging results of liver sinusoidal endothelial cells stained with fluorophores emitting at visible and near-infrared wavelengths. Quantitative comparisons of power spectral distribution and image resolution are provided.