Light Sheet Fluorescence Microscopy Research Articles

A domed window chamber for multi-modality optical imaging.

Current dorsal skin flap window chambers with flat glass windows are compatible with optical coherence tomography (OCT) and multiphoton microscopy (MPM) imaging. However, light sheet fluorescence microscopy (LSFM) performs best with a cylindrical or spherical sample located between its two 90° objectives and when all sample materials have the same index of refraction (n). A modified window chamber with a domed viewing window made from fluorinated ethylene propylene (FEP), with n similar to water and tissue, was designed. In vitro imaging of collagen gels and microsphere phantoms with and without the dome showed small decreases in signal strength and image resolution due to the dome. Using a custom mouse platform for stabilization and anesthesia support, in vivo multimodality imaging with OCT, MPM, and LSFI was demonstrated.

Increasing the acquisition speed in oblique plane microscopy via Aliasing.

Oblique plane microcopy (OPM), a variant of light-sheet fluorescence microscopy (LSFM), enables rapid volumetric imaging without mechanically scanning the sample or an objective. In an OPM, the sample space is mapped to a distortion free image space via remote focusing, and the oblique light-sheet plane is mapped via a tilted tertiary imaging system onto a camera. As a result, the 3D point-spread function and optical transfer function are tilted to the optical axis of the tertiary imaging system. To satisfy Nyquist sampling, small scanning steps are required to encompass the tilted 3D OTF, slowing down acquisition and increasing sample exposure. Here we show that a judicious amount of under-sampling can lead to a form of aliasing in OPM that can be recovered without a loss of spatial resolution or introducing artifacts. The resulting speed gain depends on the optical parameters of the system and can reach 2-4-fold in our demonstrations. We leverage this speed gain for rapid subcellular 3D imaging of mitochondrial dynamics.

Advanced Imaging Techniques for Atherosclerosis and Cardiovascular Calcification in Animal Models.

The detection and assessment of atherosclerosis and cardiovascular calcification can inform risk stratification and therapies to reduce cardiovascular morbidity and mortality. In this review, we provide an overview of current and emerging imaging techniques for assessing atherosclerosis and cardiovascular calcification in animal models. Traditional imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), offer non-invasive approaches of visualizing atherosclerotic calcification in vivo; integration of these techniques with positron emission tomography (PET) imaging adds molecular imaging capabilities, such as detection of metabolically active microcalcifications with 18F-sodium fluoride. Photoacoustic imaging provides high contrast that enables in vivo evaluation of plaque composition, yet this method is limited by optical penetration depth. Light-sheet fluorescence microscopy provides high-resolution, three-dimensional imaging of cardiovascular structures and has been used for ex vivo assessment of atherosclerotic calcification, but its limited tissue penetration and requisite complex sample preparation preclude its use in vivo to evaluate cardiac tissue. Overall, with these evolving imaging tools, our understanding of cardiovascular calcification development in animal models is improving, and the combination of traditional imaging techniques with emerging molecular imaging modalities will enhance our ability to investigate therapeutic strategies for atherosclerotic calcification.

Impact of noradrenergic inhibition on neuroinflammation and pathophysiology in mouse models of Alzheimer’s disease

Norepinephrine (NE) modulates cognitive function, arousal, attention, and responses to novelty and stress, and it also regulates neuroinflammation. We previously demonstrated behavioral and immunomodulatory effects of beta-adrenergic pharmacology in mouse models of Alzheimer’s disease (AD). The current studies were designed to block noradrenergic signaling in 5XFAD mice through (1) chemogenetic inhibition of the locus coeruleus (LC), (2) pharmacologic blocking of β-adrenergic receptors, and (3) conditional deletion of β1- or β2-adrenergic receptors (adrb1 or adrb2) in microglia.First, brain-wide AD pathology was mapped in 3D by imaging immunolabeled, cleared 5XFAD brains to assess the overlap between amyloid beta (Aβ) pathology, reactive microglia, and the loss of tyrosine hydroxylase (TH) expression in the catecholaminergic system. To examine the effects of inhibiting the LC NE system in the 5XFAD model, inhibitory (Gi) DREADD receptors were expressed specifically in LC NE neurons. LC NE neurons were chronically inhibited through the subcutaneous pump administration of the DREADD agonist clozapine-N-oxide (CNO). Plasma and brains were collected for assessment of neuroinflammation and pathology. A separate cohort of 5XFAD mice was chronically dosed with the beta-adrenergic antagonist propranolol or vehicle and evaluated for behavior, as well as post-mortem neuroinflammation and pathology. Finally, we used 5XFAD mice with conditional deletion of either adrb1 or adrb2 in microglia to assess neuroinflammation and pathology mediated by β-adrenergic signaling.Using iDISCO+, light sheet fluorescence microscopy, and novel analyses, we detected widespread microgliosis and Aβ pathology, along with modest TH downregulation in fibers across multiple brain regions, in contrast to the spatially limited TH downregulation observed in neurons. Both chemogenetic inhibition of LC adrenergic signaling and pharmacological inhibition of beta-adrenergic receptors potentiated neuroinflammation without altering Aβ pathology. Conditional deletion of adrb1 in microglia did not affect neuroinflammation. Conditional deletion of adrb2 in microglia attenuated inflammation and pathology in females but had no effect in males. Overall, these data support previous observations demonstrating the immunomodulatory effects of beta-adrenergic signaling in the pathophysiology of brain disorders and suggest that adrenergic receptors on cell types other than microglia, such as astrocytes, may mediate some of the disease-modifying effects of β-adrenergic agonists in the brain.

A Feasibility Study on 3-D Imaging of Intrahepatic Bile Ducts in Patients with Biliary Atresia Using Airy Beam Excited Two-Photon Microscopy.

The purpose of this study was to utilize a two-photon microscope excitation Airy beam to achieve three-dimensional imaging of intrahepatic bile ducts in BDL mice and patients with biliary atresia (BA). Ten male Balb/c mice aged 6-8weeks underwent extrahepatic bile duct ligation (BDL), and 10 underwent sham operation as control. After the operation, the mice resulted in symptoms such as jaundice, darkened urine, and weight loss. Taken liver tissues from BDL and control mice and trimmed to 5*5*3 mm3 after 10days. Sixteen patients with BA were included in this study. Liver transplantation was performed in 12 cases of them; liver hilar and liver margin tissues were taken during the operation. Kasai portoenterostomy (KPE) was performed in 4 cases, and liver margin tissues were taken. Intraoperative liver tissue samples were trimmed to a size of 5*5*5 mm3. The specimens were subjected to tissue fixation, antigen retrieval, antibody incubation, and subsequent tissue hyalinization following the principles of immunofluorescence staining. Subsequently, light-sheet fluorescence microscopy (LSFM) was followed, and intrahepatic bile ducts of the specimen were imaged utilizing Airy beam which was excited with high imaging depth attenuation-compensated two-photon. Deconvolution was applied to image processing to construct a three-dimensional model of intrahepatic bile ducts. Three-dimensional imaging of liver tissue was conducted in both BDL mice and BA patients, and the distribution of intrahepatic bile ducts was visualized. BDL mice exhibited notable widening of intrahepatic bile ducts, accompanied by bile duct hyperplasia. There was no obvious hyperplasia of intrahepatic bile duct in the control group. Significant small bile duct hyperplasia was seen on imaging of the intrahepatic bile ducts in patients with BA. The intrahepatic bile duct was disorganized and hyperplasia especially in patients who performed liver transplantation. The technique of Airy beam three-dimensional reconstruction can effectively image the intrahepatic bile ducts in Balb/c mice and BA patients in three dimensions. This approach contributes to a better understanding of the distribution of intrahepatic bile ducts in BA patients. Moreover, it facilitates the exploration of models that more accurately simulate BA disease by elucidating the distribution of intrahepatic bile ducts in animal models. Understanding the distribution characteristics of intrahepatic bile duct will facilitate the formulation of hilar bile duct microstructure classification, which can guide the operation and evaluate the prognosis better.

How Tumors Affect Hemodynamics: A Diffusion Study on the Zebrafish Transplantable Model of Medullary Thyroid Carcinoma by Selective Plane Illumination Microscopy.

Medullary thyroid carcinoma (MTC), a rare neuroendocrine tumor comprising 3-5% of thyroid cancers, arises from calcitonin-producing parafollicular C cells. Despite aggressive behavior, surgery remains the primary curative treatment, with limited efficacy reported for radiotherapy and chemotherapy. Recent efforts have explored the pathogenetic mechanisms of MTC, identifying it as a highly vascularized neoplasm overexpressing pro-angiogenic factors. Building on the established benefits of zebrafish embryos, we previously created an in vivo MTC xenograft platform that allows real-time observation of tumor-induced angiogenesis and evaluation of the anti-angiogenic effects of tyrosine kinase inhibitors. In this study, we present a method using selective plane illumination microscopy (SPIM) to characterize vascular permeability in these xenografted embryos. Taking advantage of dextran injections into the blood flow of zebrafish embryos, we found that the diffusion coefficient in embryos grafted with MTC cells was about tenfold lower compared with the same parameter in controls. The results demonstrate the potential of our approach to estimate diffusion parameters, providing valuable insights into vascular permeability changes in MTC-implanted zebrafish embryos compared with controls. Our study sheds light on the intricate vascular biology of MTC, offering a promising tool for future investigations into tumor-induced angiogenesis and therapeutic strategies in diverse neoplasms.

Active Remote Focus Stabilization in Oblique Plane Microscopy.

Light-sheet fluorescence microscopy (LSFM) has demonstrated great potential in the life sciences owing to its efficient volumetric imaging capabilities. For long term imaging, the light-sheet typically needs to be stabilized to the detection focal plane for the best imaging results. Current light-sheet stabilization methods rely on fluorescence emission from the sample, which may interrupt the scientific imaging and add to sample photobleaching. Here we show that for oblique plane microscopes (OPM), a subset of LSFM where a single primary objective is used for illumination and detection, light-sheet stabilization can be achieved without expending sample fluorescence. Our method achieves ~43nm axial precision and maintains the light-sheet well within the depth of focus of the detection system for hour-long acquisition runs in a lab environment that would otherwise detune the system. We demonstrate subcellular imaging of the actin skeleton in melanoma cancer cells with a stabilized OPM.

Sex-and Stress-Dependent Plasticity of a Corticotropin Releasing Hormone / GABA Projection from the Basolateral Amygdala to Nucleus Accumbens that Mediates Reward Behaviors.

Motivated behaviors are executed by refined brain circuits. Early-life adversity (ELA) is a risk for human affective disorders involving dysregulated reward behaviors. In mice, ELA causes anhedonia-like behaviors in males and augmented reward motivation in females, indicating sex-dependent disruption of reward circuit operations. We recently identified a corticotropin-releasing hormone (CRH) expressing GABAergic projection from basolateral amygdala (BLA) to nucleus accumbens (NAc) that governs reward-seeking deficits in adult ELA males, but not females. To probe the sex-specific role of this projection in reward behaviors, adult male and female CRH-Cre mice raised in control or ELA conditions received excitatory or inhibitory Cre-dependent DREADDs in BLA, and then clozapine N-oxide or vehicle to NAc medial shell during reward behaviors. We determined the cell identity of the projection using immunostaining and electrophysiology. Using tissue clearing, light sheet fluorescence microscopy and deep learning pipelines, we mapped brain-wide BLA CRH+ axonal projections to uncover sex differences in innervation. Chemogenetic manipulations in male mice demonstrated inhibitory effects of the CRH+ BLA-NAc projection on reward behaviors, whereas neither excitation nor inhibition influenced female behaviors. Molecular and electrophysiological cell-identities of the projection did not vary by sex. By contrast, comprehensive whole-brain mapping uncovered significant differences in NAc innervation patterns that were both sex and ELA-dependent, as well as selective changes of innervation of other brain regions. The CRH/GABA BLA-NAc projection that influences reward behaviors in males differs structurally and functionally in females, uncovering potential mechanisms for the profound sex-specific impacts of ELA on reward behaviors.

Imaging human pancreatic endocrinogenesis during early prenatal life.

Murine pancreatic endocrinogenesis has been extensively studied, but human data remain scarce due to limited sample availability. Here, we first built a large collection of human embryonic and fetal pancreases covering the first trimester of pregnancy to explore human endocrinogenesis. Using an experimental pipeline combining in toto staining, tissue clearing, and light-sheet fluorescence microscopy, we show that insulin+, glucagon+, and somatostatin+ cells appear simultaneously at Carnegie Stage (CS) 16. This contrasts with rodents where glucagon+ cells appear first, followed by insulin+ and finally somatostatin+ cells and highlights interspecies differences. We also detected bi-hormonal endocrine cells in 7 out of 9 human pancreases between CS16-18, which were no longer detected at later stages. We observed that cell distribution within human fetal islets resembles adult mouse islets, with a core of beta cells surrounded by alpha and delta cells, differing from a more complex arrangement in adult human islets. This, in connection with the small size of human fetal islets when compared to adult, suggests that adult human islets may form by fusion of pre-existing islets, in contrast to the mouse fission model. Together, our study provides a detailed and comprehensive description of the spatiotemporal dynamics of human pancreatic endocrinogenesis.

LungVis 1.0: an automatic AI-powered 3D imaging ecosystem unveils spatial profiling of nanoparticle delivery and acinar migration of lung macrophages

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity.

Classification of psychedelics and psychoactive drugs based on brain-wide imaging of cellular c-Fos expression.

Psilocybin, ketamine, and MDMA are psychoactive compounds that exert behavioral effects with distinguishable but also overlapping features. The growing interest in using these compounds as therapeutics necessitates preclinical assays that can accurately screen psychedelics and related analogs. We posit that a promising approach may be to measure drug action on markers of neural plasticity in native brain tissues. We therefore developed a pipeline for drug classification using light sheet fluorescence microscopy of immediate early gene expression at cellular resolution followed by machine learning. We tested male and female mice with a panel of drugs, including psilocybin, ketamine, 5-MeO-DMT, 6-fluoro-DET, MDMA, acute fluoxetine, chronic fluoxetine, and vehicle. In one-versus-rest classification, the exact drug was identified with 67% accuracy, significantly above the chance level of 12.5%. In one-versus-one classifications, psilocybin was discriminated from 5-MeO-DMT, ketamine, MDMA, or acute fluoxetine with >95% accuracy. We used Shapley additive explanation to pinpoint the brain regions driving the machine learning predictions. Our results support a novel approach for characterizing and validating psychoactive drugs with psychedelic properties.

Precise 3D Localization of Intracerebral Implants Using a Simple Brain Clearing Method.

Precise localization of intracerebral implants in rodent brains is required for physiological and behavioral studies, particularly if targeting deep brain nuclei. Traditional histological methods, based on manual estimation through sectioning can introduce errors and complicate data interpretation. Here, we introduce an alternative method based on recent advances in tissue-clearing techniques and light-sheet fluorescence microscopy. This method uses a simplified recipe of the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational Analysis (CUBIC) method, which is a rapid clearing procedure using an aqueous-based solution compatible with fluorescence and fluorescence markers. We demonstrate the utility of this approach in anesthetized transgenic mice expressing channelrhodopsin-2 (ChR2) and enhanced yellow fluorescent fusion (EYFP) protein under the choline acetyltransferase (ChAT) promoter/enhancer regions (ChAT-ChR2-EYFP mice) with implanted linear silicon optrode probes into the midbrain interpeduncular nucleus (IPN). By applying the red fluorescent DiD' dye (DiIC18(5) solid (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt) to the electrode surface, we precisely visualize the electrode localization in the IPN of ChAT-ChR2-EYFP mice. Three-dimensional brain videos from different orientations highlight the potential of this method. Optogenetic responses recorded from electrodes placed in the IPN validate these findings. This method allows for precise localization of brain implantation sites in transgenic mice expressing cell-specific fluorescence markers. It enables virtual brain slicing in any orientation, making it a useful tool for functional studies in mice.

Collagen crosslinking-induced corneal morphological changes: a three-dimensional light sheet Microscopy-based evaluation

Stiffness-related eye diseases such as keratoconus require comprehensive visualization of the complex morphological matrix changes. The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze unlabeled corneal tissue samples, qualitatively visualizing changes in corneal stiffness. Isolated porcine corneal tissue samples were treated with either NaCl or 0.1% glutaraldehyde (GTA) prior to clearing with benzyl alcohol/benzyl benzoate (BABB) and subsequently scanned with LSFM. After analysis of the LSFM data sets, the samples were embedded in paraffin to validate the results by conventional planar microscopy. In the unlabeled corneal tissue samples the 2D/3D morphology of the entire tissue volume was identified by specific autofluorescence signals. An enhancement of collagen crosslinking was induced by applying GTA to the corneal tissue. Subsequent LSFM scans showed specific morphological changes due to altered autofluorescence signals of the corneal stroma, which were confirmed by conventional histology. Therefore, LSFM analysis of corneal tissue samples allowed label-free 3D autofluorescence assessment of the corneal morphology in its anatomical context. It provides the technical basis for the examination of the pathologically altered cornea and facilitates ophthalmologic examinations of corneal diseases based on the altered tissue stiffness.

Novel application of metabolic imaging of early embryos using a light-sheet on-a-chip device: a proof-of-concept study.

Is it feasible to safely determine metabolic imaging signatures of nicotinamide adenine dinucleotide [NAD(P)H] associated auto-fluorescence in early embryos using a light-sheet on-a-chip approach? We developed an optofluidic device capable of obtaining high-resolution 3D images of the NAD(P)H autofluorescence of live mouse embryos using a light-sheet on-a-chip device as a proof-of-concept. Selecting the most suitable embryos for implantation and subsequent healthy live birth is crucial to the success rate of assisted reproduction and offspring health. Besides morphological evaluation using optical microscopy, a promising alternative is the non-invasive imaging of live embryos to establish metabolic activity performance. Indeed, in recent years, metabolic imaging has been investigated using highly advanced microscopy technologies such as fluorescence-lifetime imaging and hyperspectral microscopy. The potential safety of the system was investigated by assessing the development and viability of live embryos after embryo culture for 67 h post metabolic imaging at the two-cell embryo stage (n = 115), including a control for culture conditions and sham controls (system non-illuminated). Embryo quality of developed blastocysts was assessed by immunocytochemistry to quantify trophectoderm and inner mass cells (n = 75). Furthermore, inhibition of metabolic activity (FK866 inhibitor) during embryo culture was also assessed (n = 18). The microstructures were fabricated following a standard UV-photolithography process integrating light-sheet fluorescence microscopy into a microfluidic system, including on-chip micro-lenses to generate a light-sheet at the centre of a microchannel. Super-ovulated F1 (CBA/C57Bl6) mice were used to produce two-cell embryos and embryo culture experiments. Blastocyst formation rates and embryo quality (immunocytochemistry) were compared between the study groups. A convolutional neural network (ResNet 34) model using metabolic images was also trained. The optofluidic device was capable of obtaining high-resolution 3D images of live mouse embryos that can be linked to their metabolic activity. The system's design allowed continuous tracking of the embryo location, including high control displacement through the light-sheet and fast imaging of the embryos (<2 s), while keeping a low dose of light exposure (16 J · cm-2 and 8 J · cm-2). Optimum settings for keeping sample viability showed that a modest light dosage was capable of obtaining 30 times higher signal-noise-ratio images than images obtained with a confocal system (P < 0.00001; t-test). The results showed no significant differences between the control, illuminated and non-illuminated embryos (sham control) for embryo development as well as embryo quality at the blastocyst stage (P > 0.05; Yate's chi-squared test). Additionally, embryos with inhibited metabolic activity showed a decreased blastocyst formation rate of 22.2% compared to controls, as well as a 47% reduction in metabolic activity measured by metabolic imaging (P < 0.0001; t-test). This indicates that the optofluidic device was capable of producing metabolic images of live embryos by measuring NAD(P)H autofluorescence, allowing a novel and affordable approach. The obtained metabolic images of two-cell embryos predicted blastocyst formation with an AUC of 0.974. N/A. The study was conducted using a mouse model focused on early embryo development assessing illumination at the two-cell stage. Further safety studies are required to assess the safety and use of 405 nm light at the blastocyst stage by investigating any potential negative impact on live birth rates, offspring health, aneuploidy rates, mutational load, changes in gene expression, and/or effects on epigenome stability in newborns. This light-sheet on-a-chip approach is novel and after rigorous safety studies and a roadmap for technology development, potential future applications could be developed for ART. The overall cost-efficient fabrication of the device will facilitate scalability and integration into future devices if full-safety application is demonstrated. This work was partially supported by an Ideas Grant (no 2004126) from the National Health and Medical Research Council (NHMRC), by the Education Program in Reproduction and Development (EPRD), Department Obstetrics and Gynaecology, Monash University, and by the Department of Mechanical and Aerospace Engineering, Faculty of Engineering, Monash University. The authors E.V-O, R.N., V.J.C., A.N., and F.H. have applied for a patent on the topic of this technology (PCT/AU2023/051132). The remaining authors have nothing to disclose.

Mesoscopic axially swept oblique plane microscope for the imaging of freely moving organisms with near-isotropic resolution.

Rapid three-dimensional imaging over extended fields of view (FOVs) is crucial to the study of organism-wide systems and biological processes in vivo. Selective-plane illumination microscopy (SPIM) is a powerful method for high spatio-temporal resolution in toto imaging of such biological specimens. However, typical SPIM implementations preclude conventional sample mounting and have anisotropic imaging performance, in particular when designed for large FOVs over 1 mm diameter. Here, we introduce axial sweeping of the illumination into a non-orthogonal dual-objective oblique plane microscope (OPM) design, thereby enabling the observation of freely moving animals over millimeter-sized FOVs, at close to isotropic, sub-cellular resolution. We apply our mesoscopic axially swept OPM (MASOPM) to image the behavioral dynamics of the sea anemone Nematostella vectensis over 1 × 0.7 × 0.4 mm at 1.7 × 2.6 × 3.7 µm resolution and 0.5 Hz volume rate.

Impact of noradrenergic inhibition on neuroinflammation and pathophysiology in mouse models of Alzheimer's disease.

Norepinephrine (NE) modulates cognitive function, arousal, attention, and responses to novelty and stress, and also regulates neuroinflammation. We previously demonstrated behavioral and immunomodulatory effects of beta-adrenergic pharmacology in mouse models of Alzheimer's disease (AD). The current studies were designed to block noradrenergic signaling in 5XFAD mice through 1) chemogenetic inhibition of the locus coeruleus (LC), 2)pharmacologic blocking of β-adrenergic receptors, and 3) conditional deletion of β1- or β2-adrenergic receptors (adrb1 or adrb2) in microglia. First, brain-wide AD pathology was mapped in 3D by imaging immunolabeled, cleared 5XFAD brains to assess the overlap between Aβ pathology, reactive microglia, and the loss of tyrosine hydroxylase (TH) expression in the catecholaminergic system. To examine the effects of inhibiting the LC NE system in the 5XFAD model, inhibitory (Gi) DREADD receptors were expressed specifically in LC NE neurons. LC NE neurons were chronically inhibited through the subcutaneous pump administration of the DREADD agonist clozapine-N-oxide (CNO). Plasma and brains were collected for assessment of neuroinflammation and pathology. A separate cohort of 5XFAD mice was chronically dosed with the beta-adrenergic antagonist propranolol or vehicle and evaluated for behavior, as well as post-mortem neuroinflammation and pathology. Finally, we used 5XFAD mice with conditional deletion of either adrb1 or adrb2 in microglia to assess neuroinflammation and pathology mediated by β-adrenergic signaling. Using iDISCO, light sheet fluorescence microscopy, and novel analyses, we detected widespread microgliosis and amyloid pathology, along with modest TH downregulation in fibers across multiple brain regions, in contrast to the spatially limited TH downregulation observed in neurons. Both chemogenetic inhibition of LC adrenergic signaling and pharmacological inhibition of beta-adrenergic receptors potentiated neuroinflammation without altering amyloid beta pathology. Conditional deletion of adrb1 in microglia did not affect neuroinflammation. Conditional deletion of adrb2 in microglia attenuated inflammation and pathology in females but had no effect in males. Overall, these data support previous observations demonstrating the immunomodulatory effects of beta-adrenergic signaling in the pathophysiology of brain disorders and suggest that adrenergic receptors on cell types other than microglia, such as astrocytes, may predominantly mediate the disease-modifying effects of β-adrenergic agonists in the brain.

Rapid Whole‐Organ Characterization via Quantitative Light‐Sheet Microscopy

AbstractWhole‐organ imaging and characterization at a submicron level provide abundant information on development and diseases while remaining a big challenge, especially in the context of time load. Herein, a quantitative light‐sheet microscopy platform that enabled highly time‐efficient assessments of fibrous structures within the intact cleared tissue is developed. Dual‐view inverted selective plane illumination microscopy (diSPIM), followed by improved registration and deconvolution, led to submicron isotropic imaging of mouse upper genital tract with one hundred‐fold speed‐ups than previous efforts. Further, optical metrics quantifying 3D local density and structural complexity of targets based on parallel and vectorized convolution in both spatial and frequency domains are developed. Collectively, ≈400–2000 fold increases in time efficiency counting for imaging, postprocessing, and quantitative characterization compared to the traditional method is gained. Using this platform, automatic identification of medulla and cortex within the mouse ovary at over 90% overlap with manual selection by anatomy experts is achieved. Additionally, heterogeneous distributions of immune cells in the mouse ovary and fallopian tube, offering a unique perspective for understanding the immune microenvironment are discovered. This work paves the way for future whole‐organ study, and exhibits potential with promise for offering mechanistic insights into physiological and pathological alterations of biological tissues.

Developmental mouse brain common coordinate framework

3D brain atlases are key resources to understand the brain’s spatial organization and promote interoperability across different studies. However, unlike the adult mouse brain, the lack of developing mouse brain 3D reference atlases hinders advancements in understanding brain development. Here, we present a 3D developmental common coordinate framework (DevCCF) spanning embryonic day (E)11.5, E13.5, E15.5, E18.5, and postnatal day (P)4, P14, and P56, featuring undistorted morphologically averaged atlas templates created from magnetic resonance imaging and co-registered high-resolution light sheet fluorescence microscopy templates. The DevCCF with 3D anatomical segmentations can be downloaded or explored via an interactive 3D web-visualizer. As a use case, we utilize the DevCCF to unveil GABAergic neuron emergence in embryonic brains. Moreover, we map the Allen CCFv3 and spatial transcriptome cell-type data to our stereotaxic P56 atlas. In summary, the DevCCF is an openly accessible resource for multi-study data integration to advance our understanding of brain development.

LiveLattice: Real-time visualisation of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), move the sample substrate at an oblique angle relative to the detection objective's optical axis. Data from such tilted-sample-scan LSFMs require subsequent deskewing and rotation for proper visualisation and analysis. Such data preprocessing operations currently demand substantial memory allocation and pose significant computational challenges for large 4D dataset. The consequence is prolonged data preprocessing time compared to data acquisition time, which limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a memory-efficient transformation algorithm for deskewing and rotating the raw dataset, significantly reducing memory usage and the run time by more than 10-fold for large image stacks. Benchmarked against the conventional method and existing software, our approach demonstrates linear runtime compared to the cubic and quadratic runtime of the other approaches. Preprocessing a raw 3D volume of 2 GB (512 × 1536 × 600 pixels) can be accomplished in 3 s using a GPU with 24 GB of memory on a single workstation. Applied to 4D LLSM datasets of human hepatocytes, lung organoid tissue and brain organoid tissue, our method provided rapid and accurate preprocessing within seconds. Importantly, such preprocessing speeds now allow visualisation of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data preprocessing, visualisation, and analysis on standard workstations, thereby revolutionising biological imaging applications for LLSM and similar microscopes.

Unlocking the potential of large-scale 3D imaging with tissue clearing techniques.

The three-dimensional (3D) anatomical structure of living organisms is intrinsically linked to their functions, yet modern life sciences have not fully explored this aspect. Recently, the combination of efficient tissue clearing techniques and light-sheet fluorescence microscopy (LSFM) for rapid 3D imaging has improved access to 3D spatial information in biological systems. This technology has found applications in various fields, including neuroscience, cancer research, and clinical histopathology, leading to significant insights. It allows imaging of entire organs or even whole bodies of animals and humans at multiple scales. Moreover, it enables a form of spatial omics by capturing and analyzing cellome information, which represents the complete spatial organization of cells. While current 3D imaging of cleared tissues has limitations in obtaining sufficient molecular information, emerging technologies such as multi-round tissue staining and super-multicolor imaging are expected to address these constraints. 3D imaging using tissue clearing and light-sheet microscopy thus offers a valuable research tool in the current and future life sciences for acquiring and analyzing large-scale biological spatial information.