ABSTRACT Speckled illumination enhances widefield fluorescence microscopy by enabling optical sectioning and super resolution. In random illumination microscopy, sequences of speckled illumination patterns are used to excite fluorescent samples and images are reconstructed based on a statistical analysis of the intensity fluctuations. Although random illumination microscopy has been shown to give excellent performance, its widespread implementation is hindered by the high cost and complexity of the generation of suitable speckled illumination patterns, which is typically achieved using digital micro‐mirror devices or spatial light modulators. Here, we present a zwitterion‐doped liquid crystal device capable of generating independent, high‐contrast speckle patterns with a tunable decorrelation time in the 0.1 s – 0.1 ms range under visible laser illumination. This liquid crystal based dynamic speckle generator is applied to widefield random illumination fluorescence microscopy of tissue and cell samples, where it enables optical sectioning with a 2 axial resolution, and a 1.5 ‐ fold improvement in lateral spatial resolution. Owing to its low cost and simplicity, this liquid crystal speckle generator offers an attractive alternative to digital micro‐mirror and spatial light modulator devices for implementing widefield random illumination microscopy.

Optical sectioning in wide-field two-photon microscopy using temporal focusing and random illumination

Primary cilia are non-motile sensory organelles that detect extracellular signals; disruptions in their functions are linked to neurodevelopmental disorders. Because cilia lengths can rapidly change in response to stressors, they are important for both plasticity and brain homeostasis. Accurate measurement of ciliary length is therefore essential but the absence of standardized methods and the variability introduced by different techniques can compromise measurement reliability and precision. To address this challenge, our study employed two distinct methods to estimate the length of primary cilia in hippocampal subregions in mice. We compared stereology-based 3D quantification, which is considered a methodological gold standard due to its unbiased sampling design and correction for tissue shrinkage, with 3D reconstruction to measure primary cilia length. 3D reconstruction imaging used a 100× oil-immersion objective. With neuronal cilia typically ∼2-10 µm long in hippocampus, a 1-µm z-step provided multiple optical sections per cilium thereby ensuring full structural visualization. Our findings show that both methods allow simple and equally precise measurements of neuronal primary cilia length in hippocampal subregions. Their strong agreement provides researchers with reliable tools for studying primary cilia on immunohistochemically stained sections and supports a consistent methodological framework for investigating cilia dynamics.

We describe a novel method for adapting a two-photon scanning microscope to enable simultaneous detection of two-photon generated visible fluorescence and single-photon generated near-infrared (nIR) fluorescence. In this configuration, nIR fluorescence is routed through a single-mode optical fiber before detection by a photomultiplier tube. This fiber coupling offers two advantages: first, the optical fiber functions as a pinhole aperture, allowing for improved optical sectioning of the nIR signal; second, it minimizes nIR background fluorescence. To validate the effectiveness of this design, we conducted two sets of experiments. First, we compare two fluorescence indicators of the neurotransmitter dopamine: the genetically encoded indicator GRABDA and single walled carbon nanotube based optical nanosensors (nIRCats). Although nIRCats exhibit lower affinity for dopamine than GRABDA, this property allows for identification of high concentration release sites in the striatum. Second, we simultaneously imaged depolarization-induced calcium changes and dopamine release in the retina. Together, these results demonstrate the utility of integrating confocal nIR detection into a two-photon platform for simultaneous functional imaging across complementary spectral channels.

Tunneling nanotubes (TNTs) are thin, actin-based intercellular conduits that enable long-range transfer of organelles and signaling cargo. Although widely reported across multiple cell types, their presence in human skin cells has not been well described. This article describes a standardized protocol to detect and characterize TNTs in vitro between human epidermal keratinocytes and dermal fibroblasts. The method involves preparing a co-culture of primary cells, gentle fixation to preserve fragile TNTs, membrane labeling with wheat germ agglutinin, F-actin staining with phalloidin, and systematic z-stack imaging by inverted confocal microscopy to distinguish TNTs suspended above the substratum from adherent filopodia. Optional immunostaining for α-tubulin allows assessment of microtubule incorporation. TNTs are defined by three features: thin, straight protrusions connecting two or more cells, the presence of F-actin, and continuity across cell pairs in serial optical sections. Representative results demonstrate TNTs linking dermal-dermal, epidermal-epidermal, and dermal-epidermal pairs, with variable cytoskeletal composition (F-actin alone or F-actin plus α-tubulin). Critical steps include gentle fixation, use of fresh reagents, and acquisition of sufficient z-planes to avoid misclassification, while common artifacts include TNT breakage and incomplete staining. Together, these optimized steps enable reproducible TNT detection in skin cell systems and offer a methodological basis for future investigation of TNT-mediated communication in skin biology and regeneration.

The importance of optical coherence tomographic sectioning in a case with unilateral subtle visual disturbance related to a vertical intraretinal hyperreflective line

Androgen receptor blockade and its effect on PSMA-localization in prostate cancer: Implications for radioligand therapy.

The distribution of seepage field in embankment dams is an important aspect of the safe operation of in-service embankment dams. The distributed optical fiber temperature monitoring technology has some advantages of high sensitivity, strong real-time performance, and rich data. This is a problem worthy of study for the monitoring of seepage field in embankment dams. This paper takes a certain embankment dam as an example. It sets up some optical fiber temperature measurement sections near the traditional seepage monitoring section. It elaborately introduces the optical fiber layout, on-site construction, long-term monitoring, and simulation. The result shows that the position of the infiltration line can be measured by using heated distributed optical fibers; the error is within the range of 0.1 to 0.2 m. The monitoring results are basically consistent with the traditional seepage monitoring results, indicating that it is feasible to use distributed optical fiber temperature measurement technology for dam seepage monitoring. Long-term monitoring and numerical simulation have obtained the infiltration lines, velocity vectors, and streamlines at different water levels, verifying the reliability of the distributed optical fiber temperature monitoring technology. As summarized, the distributed optical fiber temperature measurement technology can accurately obtain the seepage information inside the dam body, providing a new idea for the analysis and safety assessment of the seepage field of embankment dams.

Structured illumination microscopy (SIM) is a powerful method for fast and gentle live-cell super-resolution imaging. However, its susceptibility to reconstruction artifacts from out-of-focus blur and background imposes substantial barriers to analyze the dynamics of densely packed volumetric intraorganellar ultrastructures that are typically in a size range of SIM’s spatial resolution. To address this limitation, we have developed Lock-in-SIM, an open-access two-dimensional SIM framework that eliminates background and maximizes the recovery of sub-diffraction information with the highest possible frequency extraction. By leveraging the intrinsic modulation differences of volumetric sample structures, Lock-in-SIM enables efficient optical sectioning, extends imaging depth, and improves data fidelity and quantifiability. We demonstrate the superiority of Lock-in-SIM by visualizing various challenging intraorganellar ultrastructures in live cells. Our investigations uncover mechanisms of mitochondrial fission and endoplasmic reticulum-lysosome interactions and provide insights into the intricate yet highly regulated structural remodeling of organelles.

Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35°, the median bead FWHM for 100 nm diameter fluorescent beads in x, y, and z and the optical sectioning strength were measured over a volume of 100 × 100 × 100 μm3 to be 0.29, 0.31, 0.83, and 2.45–3.00 μm, respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multifield-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose–response curves in spheroids. We also use it to perform time-lapse multifield-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.

Spinning disk confocal microscopy enables fast optical sectioning with low phototoxicity but is often inaccessible due to high hardware costs. We present a lower-cost solution using a 25-megapixel machine vision CMOS camera and a custom-built spinning disk. This camera uses a back-illuminated sensor with high quantum efficiency and low read noise. High-resolution images of Thy1-GFP mouse brain slices, Drosophila embryos and larvae, and H&E-stained rat testis verified performance across 3D tissue volumes. The measured resolution was 215.8 nm in X, Y and 521.9 nm in Z with a 60×/1.42 NA objective. The custom disk, made with 18 µm pinholes (180 µm pitch) on a chrome photomask and mounted to an optical chopper motor, enables stable, near-telecentric imaging at lower magnifications. Micromanager software integration allows synchronized control of all hardware, which demonstrates that affordable CMOS sensors can potentially replace sCMOS in spinning disk microscopy, offering an open-access, scalable solution for advanced imaging.

Fresnel incoherent correlation holography (FINCH) is a widely used incoherent digital holography technique. FINCH has a higher lateral resolution but a lower axial resolution compared to those of direct imaging methods with the same numerical aperture. The low axial resolution problem of FINCH was addressed by developing sectioning FINCH methods implemented by pixel-by-pixel electronic scanning of a phase pinhole to achieve sectioning capability in FINCH, mimicking a conventional confocal microscope. The above approach requires not only an additional spatial light modulator for electronic scanning but also time-consuming data acquisition and processing. In this study, for the first time, to the best of our knowledge, we developed a fully computational sectioning method for FINCH. This computational optical sectioning FINCH (COS-FINCH) exploits the axial intensity and phase characteristics of FINCH holograms and first and second order derivatives of axial intensity distributions to identify the object planes, extract information, and achieve sectioning. Extensive simulation studies and results of preliminary experimental studies are presented.

Innovations in 3D tissue imaging have revolutionized research, but limitations stemming from lengthy protocols and equipment accessibility persist. Widefield microscopy is fast and accessible but often excluded from 3D imaging workflows due to its lack of optical sectioning. Here we combine tissue clearing with a commercial depth-variant deconvolution approach that we optimized for large-volume widefield imaging. By implementing prefiltering with z-brick splitting, we achieve subnuclear axial resolution in tissues to a depth of 500 µm in multi-tile scan images. We illustrate the utility of this method in a model of ileitis and to gain a 3D perspective in thick brain slices from a model of cerebral amyloid angiopathy, where we resolve amyloid deposits along small blood vessels, attaining resolution that compared favorably to confocal microscopy. Finally, we leverage our approach to image hundreds of consecutive z planes for richer evaluation of cleared human kidney biopsies in a simulated, transplant time window and visualized atrophic tubules and winding arterioles associated with glomeruli in 3D. Having achieved subnuclear z-resolution in sections hundreds of microns thick, coupling widefield microscopy of cleared tissue to robust deconvolution now emerges as an accessible and viable method to gain 3D insight in research or clinical evaluations.

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 tradeoffs 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 × 1.3 mm2 FOV with an effective 200 μm 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.

Abstract Description The thymus, as the primary site of T cell maturation, plays a critical role in the functionality of the adaptive immune system, and as such, the recovery of the thymus after injury and the role that the thymus vasculature system plays in the thymus regeneration process is of significant medical interest. Previously, our lab developed an intravital thymus imaging method and found significant increases in thymus blood vessel diameter, leakage, frequency, and area after sublethal total body irradiation (SL-TBI). To further study hemodynamics and thymocyte motility, we developed a vacuum-stabilized two-photon microendoscope. The stabilization of the vacuum system was found to sufficiently minimize tissue movement (±5 μm displacement) and enable optical sectioning of the thymus. To study thymus recovery, wild-type (C57BL/6J) and UBC-GFP (C57BL/6-Tg (UBC-GFP)30Scha/J) mice ranging in age from 8-52 weeks received SL-TBI (4.5 Gy). Wild-type mice received 106 whole BM cells from UBC-GFP mice 1-day after SL-TBI and were imaged 7/14-days post transplantation to study thymocyte motility. In contrast to previous measurements, blood vessel diameters were found to significantly decrease from 7 to 14 days and significant thymocyte motility was observed, with a max average velocity of ∼15 μm/min. These results provide insight into the thymus recovery process and demonstrate intravital microendoscopy as a powerful tool to study the native thymus. Funding Sources NIH 1R01HL171971-01A1 Topic Categories Technological Innovations in Immunology (TECH)

Three-dimensional structured illumination microscopy (3D-SIM) doubles the resolution of fluorescence imaging in all directions and enables optical sectioning with increased image contrast. However, 3D-SIM has not been widely applied to imaging deep in thick tissues due to its sensitivity to sample-induced aberrations, making the method difficult to apply beyond 10 µm in depth. Furthermore, 3D-SIM has not been available in an upright configuration, limiting its use for live imaging while manipulating the specimen, for example, with electrophysiology. Here, we have overcome these barriers by developing a novel upright 3D-SIM system (termed Deep3DSIM) that incorporates adaptive optics for aberration correction and remote focusing, reducing artefacts, improving contrast, restoring resolution, and eliminating the need to move the specimen or the objective lens in volume imaging. These advantages are equally applicable to inverted 3D-SIM systems. We demonstrate high-quality 3D-SIM imaging in various samples, including imaging more than 130 µm into the Drosophila brain.

Super-resolution microscopy has pushed the limits of biological imaging. However, achieving isotropic resolution across all spatial dimensions remains a challenge and often requires a complex and highly sensitive optical setup. Herein, we introduce axial interference speckle illumination-engineered structured illumination microscopy (AXIS-SIM), a minimal-modification approach that utilizes constructive interference from a simple back-reflecting mirror to enhance the axial resolution without additional phase control or complex beam shaping. AXIS-SIM provides superior optical sectioning and improves axial resolution beyond the typical axial resolution of conventional 3D-structured illumination microscopy (~300 nm), achieving lateral and axial resolutions of 108.5 and 140.1 nm, respectively. Furthermore, its robustness against alignment errors and sample-induced aberrations enables high-throughput 3D super-resolution imaging of diverse biological specimens. We demonstrate its potential by visualizing the 3D morphology of cell membranes, resolving the nanoscale distribution of lysosomes and microtubules and tracking lysosomal movements with enhanced axial clarity.

Image scanning microscopy (ISM) is a promising imaging technique that offers sub-diffraction-limited resolution and optical sectioning. Theoretically, ISM can improve the optical resolution by a factor of two through pixel reassignment and deconvolution. Multifocal array illumination and scanning have been widely adopted to implement ISM because of their simplicity. Conventionally, digital micromirror devices (DMDs)1 and microlens arrays (MLAs)2,3 have been used to generate dense and uniform multifocal arrays for ISM, which are critical for achieving fast imaging and high-quality ISM reconstruction. However, these approaches have limitations in terms of cost, numerical aperture (NA), pitch, and uniformity, making it challenging to create dense and high-quality multifocal arrays at high NA. To overcome these limitations, we introduced a novel multifocal metalens design strategy called the hybrid multiplexing method, which combines two conventional multiplexing approaches: phase addition and random multiplexing. Through numerical simulations, we demonstrate that the proposed method generates more uniform and denser multifocal arrays than conventional methods, even at small pitches. As a proof of concept, we fabricated a multifocal metalens generating 40 × 40 array of foci with a 3 μm pitch and NA of 0.7 operating at a wavelength of 488 nm and then constructed the multifocal metalens-based ISM (MMISM). We demonstrated that MMISM successfully resolved sub-diffraction-limited features in imaging of microbead samples and forebrain organoid sections. The results showed that MMISM imaging achieved twice the diffraction-limited resolution and revealed clearer structural features of neurons compared to wide-field images. We anticipate that our novel design strategy can be widely applied to produce multifunctional optical elements and replace conventional optical elements in specialized applications.

Structured illumination microscopy for optical sectioning is widely used for micro- and nanoscale surface topography measurement, but the conventional phase-shifting approach requires multiple image acquisitions, leading to a trade-off between measurement speed and accuracy. To overcome this limitation, we propose a complementary binary fringe difference method for structured illumination microscopy to obtain optical sectioning at each layer. Theoretical derivation and simulation experiments demonstrate that the modulation signal obtained through complementary binary fringe difference exhibits an axial intensity profile approximating a symmetric Bessel function curve. This enables the determination of depth positions corresponding to peak intensities via Gaussian function fitting, thereby realizing surface shape measurement. Experimental results on a 1.8 µm step-height sample demonstrated the effectiveness of the proposed method, achieving a root-mean-square error of 29 nm.

Structured illumination microscopy (SIM) yields optically-sectioned images by projecting fringe patterns on a sample and laterally shifting the fringes three times. In this paper, we introduce a conditional framework entitled optically-sectioning diffusion (OSdiffuse) model for 3D microscopic imaging. This framework reconstructs a sectioned image using a single wide-field image as input. It effectively suppresses background noise similar to optical sectioning SIM (OS-SIM), but it reduces the data acquisition by threefold. Furthermore, the model enhances the axial sectioning capability twofold compared to the conventional OS-SIM approach. The effectiveness of the proposed mode was demonstrated with both simulations and experiments. We believe that the proposed method will be widely used in biological studies.