High-resolution open-top axially swept light sheet microscopy.

BACKGROUND: Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for the high-throughput cellular imaging of optically cleared, large-sized specimens, such as the brain. Despite the development of various OT-LSM techniques, achieving submicron resolution in all dimensions remains. RESULTS: We developed a high-resolution open-top axially swept LSM (HR-OTAS-LSM) for high-throughput and high-resolution imaging in all dimensions. High axial and lateral resolutions were achieved by using an aberration-corrected axially swept excitation light sheet in the illumination arm and a high numerical aperture (NA) immersion objective lens in the imaging arm, respectively. The high-resolution, high-throughput visualization of neuronal networks in mouse brain and retina specimens validated the performance of HR-OTAS-LSM. CONCLUSIONS: The proposed HR-OTAS-LSM method represents a significant advancement in the high-resolution mapping of cellular networks in biological systems such as the brain and retina.

Fast volumetric imaging with line-scan confocal microscopy by electrically tunable lens at resonant frequency.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.

Deep learning enables reference-free isotropic super-resolution for volumetric fluorescence microscopy.

Volumetric imaging by fluorescence microscopy is often limited by anisotropic spatial resolution, in which the axial resolution is inferior to the lateral resolution. To address this problem, we present a deep-learning-enabled unsupervised super-resolution technique that enhances anisotropic images in volumetric fluorescence microscopy. In contrast to the existing deep learning approaches that require matched high-resolution target images, our method greatly reduces the effort to be put into practice as the training of a network requires only a single 3D image stack, without a priori knowledge of the image formation process, registration of training data, or separate acquisition of target data. This is achieved based on the optimal transport-driven cycle-consistent generative adversarial network that learns from an unpaired matching between high-resolution 2D images in the lateral image plane and low-resolution 2D images in other planes. Using fluorescence confocal microscopy and light-sheet microscopy, we demonstrate that the trained network not only enhances axial resolution but also restores suppressed visual details between the imaging planes and removes imaging artifacts.

Optimized single-step optical clearing solution for 3D volume imaging of biological structures.

Various optical clearing approaches have been introduced to meet the growing demand for 3D volume imaging of biological structures. Each has its own strengths but still suffers from low transparency, long incubation time, processing complexity, tissue deformation, or fluorescence quenching, and a single solution that best satisfies all aspects has yet been developed. Here, we develop OptiMuS, an optimized single-step solution that overcomes the shortcomings of the existing aqueous-based clearing methods and that provides the best performance in terms of transparency, clearing rate, and size retention. OptiMuS achieves rapid and high transparency of brain tissues and other intact organs while preserving the size and fluorescent signal of the tissues. Moreover, OptiMuS is compatible with the use of lipophilic dyes, revealing DiI-labeled vascular structures of the whole brain, kidney, spleen, and intestine, and is also applied to 3D quantitative and comparative analysis of DiI-labeled vascular structures of glomeruli turfs in normal and diseased kidneys. Together, OptiMuS provides a single-step solution for simple, fast, and versatile optical clearing method to obtain high tissue transparency with minimum structural changes and is widely applicable for 3D imaging of various whole biological structures.

Sodium Cholate-Based Active Delipidation for Rapid and Efficient Clearing and Immunostaining of Deep Biological Samples.

Recent surges of optical clearing provided anatomical maps to understand structure-function relationships at organ scale. Detergent-mediated lipid removal enhances optical clearing and allows efficient penetration of antibodies inside tissues, and sodium dodecyl sulfate (SDS) is the most common choice for this purpose. SDS, however, forms large micelles and has a low critical micelle concentration (CMC). Theoretically, detergents that form smaller micelles and higher CMC should perform better but these have remained mostly unexplored. Here, SCARF, a sodium cholate (SC)-based active delipidation method, is developed for better clearing and immunolabeling of thick tissues or whole organs. It is found that SC has superior properties to SDS as a detergent but has serious problems; precipitation and browning. These limitations are overcome by using the ion-conductive film to confine SC while enabling high conductivity. SCARF renders orders of magnitude faster tissue transparency than the SDS-based method, while excellently preserving the endogenous fluorescence, and enables much efficient penetration of a range of antibodies, thus revealing structural details of various organs including sturdy post-mortem human brain tissues at the cellular resolution. Thus, SCARF represents a robust and superior alternative to the SDS-based clearing methods and is expected to facilitate the 3D morphological mapping of various organs.

Open-top axially swept light-sheet microscopy.

Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for high throughput cellular imaging of large tissue specimens including optically cleared tissues by having the entire optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with improved axial resolution. OTAS-LSM swept a tightly focused excitation light sheet across the imaging FOV using an electro tunable lens (ETL) and collected emission light at the focus of the light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a liquid prism and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment, and the image resolutions were under 1.6µm in all directions. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine, and it demonstrated the single-cell resolution imaging of neuronal networks. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues.

Rapid immunostaining method for three-dimensional volume imaging of biological tissues by magnetic force-induced focusing of the electric field.

Recent surges in tissue clearing technology have greatly advanced 3-dimensional (3D) volume imaging. Cleared tissues need to be stained with fluorescence probes for imaging but the current staining methods are too laborious and inefficient for thick 3D samples, which impedes the broad application of clearing technology. To overcome these limitations, we developed an advanced staining platform named EFIC in which a magnetic force focuses the electric field by bending it onto the sample. Such that EFIC applies a significantly lower electric field to maintain nanoscale structural integrity while effectively drives staining probes into pre-cleared 3D samples. We found that EFIC achieved a rapid and uniform staining of various proteins and vascular networks of the brain as well as other organs over the entire depth of imaging. EFIC stained tau deposits and the vascular structure in the post-mortem human brain of Alzheimer's disease and intracerebral hemorrhage, respectively, enabling quantitative analysis. The effectiveness of EFIC was further extended by the successful staining of various targets in non-cleared 3D brain samples. Together, EFIC represents a versatile and reliable staining platform for rapidly analyzing 3D molecular signatures and can be applied to sectioning-free 3D histopathology for diagnostic purposes.

Immune cell composition in normal human kidneys.

An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3+ T cells. Kidney CD4+ and CD8+ T cells comprised 44% and 56% of total T cells. Of these, 47% ± 15% of T cells displayed an effector memory phenotype (CCR7- CD45RA- CD69-), and 48% ± 19% were kidney-resident cells (CCR7- CD45RA- CD69+). However, the proportions of human CD14+ and CD16+ myeloid cells were approximately 10% of total immune cells. A predominance of CD3+ T cells and a low proportion of CD14+ or CD68+ myeloid cells were also identified in healthy human kidney sections. In mouse kidneys, kidney-resident macrophages (CD11blow F4/80high) were the most predominant subset (up to 50%) but the proportion of CD3+ T cells was less than 20%. These results will be of use in studies in which mouse results are translated into human cases under homeostatic conditions or with disease.

Activation of CaMKIV by soluble amyloid-ß1-42 impedes trafficking of axonal vesicles and impairs activity-dependent synaptogenesis.

The prefibrillar form of soluble amyloid-ß (sAß1-42) impairs synaptic function and is associated with the early phase of Alzheimer's disease (AD). We investigated how sAß1-42 led to presynaptic defects using a quantum dot-based, single particle-tracking method to monitor synaptic vesicle (SV) trafficking along axons. We found that sAß1-42 prevented new synapse formation induced by chemical long-term potentiation (cLTP). In cultured rat hippocampal neurons, nanomolar amounts of sAß1-42 impaired Ca2+ clearance from presynaptic terminals and increased the basal Ca2+ concentration. This caused an increase in the phosphorylation of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and its substrate synapsin, which markedly inhibited SV trafficking along axons between synapses. Neurons derived from a transgenic AD mouse model had similar defects, which were prevented by an inhibitor of CaMK kinase (CaMKK; which activates CaMKIV), by antibodies against Aß1-42, or by expression a phosphodeficient synapsin mutant. The CaMKK inhibitor also abolished the defects in activity-dependent synaptogenesis caused by sAß1-42 Our results suggest that by disrupting SV reallocation between synapses, sAß1-42 prevents neurons from forming new synapses or adjusting strength and activity among neighboring synapses. Targeting this mechanism might prevent synaptic dysfunction in AD patients.

nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology.

Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

SNX14 is a bifunctional negative regulator for neuronal 5-HT6 receptor signaling.

The 5-hydroxytryptamine (5-HT, also known as serotonin) subtype 6 receptor (5-HT6R, also known as HTR6) plays roles in cognition, anxiety and learning and memory disorders, yet new details concerning its regulation remain poorly understood. In this study, we found that 5-HT6R directly interacted with SNX14 and that this interaction dramatically increased internalization and degradation of 5-HT6R. Knockdown of endogenous SNX14 had the opposite effect. SNX14 is highly expressed in the brain and contains a putative regulator of G-protein signaling (RGS) domain. Although its RGS domain was found to be non-functional as a GTPase activator for Gαs, we found that it specifically bound to and sequestered Gαs, thus inhibiting downstream cAMP production. We further found that protein kinase A (PKA)-mediated phosphorylation of SNX14 inhibited its binding to Gαs and diverted SNX14 from Gαs binding to 5-HT6R binding, thus facilitating the endocytic degradation of the receptor. Therefore, our results suggest that SNX14 is a dual endogenous negative regulator in 5-HT6R-mediated signaling pathway, modulating both signaling and trafficking of 5-HT6R.