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Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal–oxide–semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.

Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are demonstrated. Images of beads prove a near-isotropic lateral resolution of sub-100 nm. Images of yeast endoplasmic reticulum show that coherent structured illumination (csi) LSFM performs with physiologically relevant specimens.

Tau pathology of the noradrenergic locus coeruleus (LC) is a hallmark of several age-related neurodegenerative disorders, including Alzheimer’s disease. However, a comprehensive neuropathological examination of the LC is difficult due to its small size and rod-like shape. To investigate the LC cytoarchitecture and tau cytoskeletal pathology in relation to possible propagation patterns of disease-associated tau in an unprecedented large-scale three-dimensional view, we utilized volume immunostaining and optical clearing technology combined with light sheet fluorescence microscopy. We examined AT8+ pathological tau in the LC/pericoerulear region of 20 brains from Braak neurofibrillary tangle (NFT) stage 0–6. We demonstrate an intriguing morphological complexity and heterogeneity of AT8+ cellular structures in the LC, representing various intracellular stages of NFT maturation and their diverse transition forms. We describe novel morphologies of neuronal tau pathology such as AT8+ cells with fine filamentous somatic protrusions or with disintegrating soma. We show that gradual dendritic atrophy is the first morphological sign of the degeneration of tangle-bearing neurons, even preceding axonal lesions. Interestingly, irrespective of the Braak NFT stage, tau pathology is more advanced in the dorsal LC that preferentially projects to vulnerable forebrain regions in Alzheimer’s disease, like the hippocampus or neocortical areas, compared to the ventral LC projecting to the cerebellum and medulla. Moreover, already in the precortical Braak 0 stage, 3D analysis reveals clustering tendency and dendro-dendritic close appositions of AT8+ LC neurons, AT8+ long axons of NFT-bearing cells that join the ascending dorsal noradrenergic bundle after leaving the LC, as well as AT8+ processes of NFT-bearing LC neurons that target the 4th ventricle wall. Our study suggests that the unique cytoarchitecture, comprised of a densely packed and dendritically extensively interconnected neuronal network with long projections, makes the human LC to be an ideal anatomical template for early accumulation and trans-neuronal spreading of hyperphosphorylated tau.

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy.

Optical microscopy has vastly expanded the frontiers of structural and functional biology, due to the non-invasive probing of dynamic volumes in vivo. However, traditional widefield microscopy illuminating the entire field of view (FOV) is adversely affected by out-of-focus light scatter. Consequently, standard upright or inverted microscopes are inept in sampling diffraction-limited volumes smaller than the optical system’s point spread function (PSF). Over the last few decades, several planar and structured (sinusoidal) illumination modalities have offered unprecedented access to sub-cellular organelles and 4D (3D + time) image acquisition. Furthermore, these optical sectioning systems remain unaffected by the size of biological samples, providing high signal-to-noise (SNR) ratios for objective lenses (OLs) with long working distances (WDs). This review aims to guide biologists regarding planar illumination strategies, capable of harnessing sub-micron spatial resolution with a millimeter depth of penetration.

Annexin 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins in plants. Generally, annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking, and stress responses. The expression of annexins is developmentally regulated, and it is sensitive to the external environment. ANN1 is expressed in almost all Arabidopsis tissues, while the most abundant is in the root, root hairs, and in the hypocotyl epidermal cells. Annexins were also occasionally proposed to associate with cytoskeleton and vesicles, but they were never developmentally localized at the subcellular level in diverse plant tissues and organs. Using advanced light-sheet fluorescence microscopy (LSFM), we followed the developmental and subcellular localization of GFP-tagged ANN1 in post-embryonic Arabidopsis organs. By contrast to conventional microscopy, LSFM allowed long-term imaging of ANN1-GFP in Arabidopsis plants at near-environmental conditions without affecting plant viability. We studied developmental regulation of ANN1-GFP expression and localization in growing Arabidopsis roots: strong accumulation was found in the root cap and epidermal cells (preferentially in elongating trichoblasts), but it was depleted in dividing cells localized in deeper layers of the root meristem. During root hair development, ANN1-GFP accumulated at the tips of emerging and growing root hairs, which was accompanied by decreased abundance in the trichoblasts. In aerial plant parts, ANN1-GFP was localized mainly in the cortical cytoplasm of trichomes and epidermal cells of hypocotyls, cotyledons, true leaves, and their petioles. At the subcellular level, ANN1-GFP was enriched at the plasma membrane (PM) and vesicles of non-dividing cells and in mitotic and cytokinetic microtubular arrays of dividing cells. Additionally, an independent immunolocalization method confirmed ANN1-GFP association with mitotic and cytokinetic microtubules (PPBs and phragmoplasts) in dividing cells of the lateral root cap. Lattice LSFM revealed subcellular accumulation of ANN1-GFP around the nuclear envelope of elongating trichoblasts. Massive relocation and accumulation of ANN1-GFP at the PM and in Hechtian strands and reticulum in plasmolyzed cells suggest a possible osmoprotective role of ANN1-GFP during plasmolysis/deplasmolysis cycle. This study shows complex developmental and subcellular localization patterns of ANN1 in living Arabidopsis plants.

Three-dimensional (3D) ex vivo imaging of cleared tissue from intact brains from animal models, human brain surgical specimens, and large postmortem human and non-human primate brain specimens is essential for understanding physiological neural connectivity and pathological alterations underlying neurological and neuropsychiatric disorders. Contemporary light-sheet microscopy enables rapid, high-resolution imaging of large, cleared samples but is limited by the orthogonal arrangement of illumination and detection optics, which constrains specimen size. Light-sheet theta microscopy (LSTM) overcomes this limitation by employing two oblique illumination paths while maintaining a perpendicular detection geometry. Here, we report the development of a next-generation, fully integrated and user-friendly LSTM system that enables uniform subcellular-resolution imaging (with subcellular resolution determined by the lateral performance of the system) throughout large specimens without constraining lateral (XY) dimensions. The system provides a seamless workflow encompassing image acquisition, data storage, pre- and post-processing, enhancement and quantitative analysis. Performance is demonstrated by high-resolution 3D imaging of intact mouse brains and human brain samples, including complete downstream analyses such as digital neuron tracing, vascular reconstruction and design-based stereological analysis. This enhanced and accessible LSTM implementation enables rapid quantitative mapping of molecular and cellular features in very large biological specimens.

Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition. Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM. Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM. Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz. Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.