Volumetric analysis of the terminal ductal lobular unit architecture and cell phenotypes in the human breast.

The major lactiferous ducts of the human breast branch out and end at terminal ductal lobular units (TDLUs). Despite their functional and clinical importance, the three-dimensional (3D) architecture of TDLUs has remained undetermined. Our quantitative and volumetric imaging of healthy human breast tissue demonstrates that highly branched TDLUs, which exhibit increased proliferation, are uncommon in the resting tissue regardless of donor age, parity, or hormonal contraception. Overall, TDLUs have a consistent shape and branch parameters, and they contain a main subtree that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. Simulation of TDLU branching morphogenesis in three dimensions suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice despite their anatomical differences. In all, our data provide structural insight into 3D anatomy and branching of the human breast and exemplify the power of volumetric imaging in gaining a deeper understanding of breast biology.

A Simple and Fast Optical Clearing Method for Whole-Mount Fluorescence In Situ Hybridization (FISH) Imaging.

We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution three-dimensional (3D) images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

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.

3D light-sheet fluorescence microscopy in preclinical and clinical drug discovery.

Light-sheet fluorescence microscopy (LSFM) combined with tissue clearing has emerged as a powerful technology in drug discovery. LSFM is applicable to a variety of samples, from rodent organs to clinical tissue biopsies, and has been used for characterizing drug targets in tissues, demonstrating the biodistribution of pharmaceuticals and determining their efficacy and mode of action. LSFM is scalable to high-throughput analysis and provides resolution down to the single cell level. In this review, we describe the advantages of implementing LSFM into the drug discovery pipeline and highlight recent advances in this field.

Delineating, Imaging, and Assessing Pulmonary Fibrosis Remodeling via Collagen Hybridization.

Idiopathic pulmonary fibrosis (IPF) is a progressive, life-threatening disease with no early detection, few treatments, and dismal outcomes. Although collagen overdeposition is a hallmark of lung fibrosis, current research mostly focuses on the cellular aspect, leaving collagen, particularly its dynamic remodeling (i.e., degradation and turnover), largely unexplored. Here, using a collagen hybridizing peptide (CHP) that specifically binds unfolded collagen chains, we reveal vast collagen denaturation in human IPF lungs and delineate the spatiotemporal progression of collagen denaturation three-dimensionally within fibrotic lungs in mice. Transcriptomic analyses support that lung collagen denaturation is strongly associated with up-regulated collagen catabolism in mice and patients. We thus show that CHP probing differentiates remodeling responses to antifibrotics and highlights the resolution of established fibrosis by agents up-regulating collagen catabolism. We further develop a radioactive CHP that detects fibrosis in vivo in mice as early as 7 days postlung-injury (Ashcroft score: 2-3) by positron emission tomography (PET) imaging and ex vivo in clinical lung specimens. These findings establish collagen denaturation as a promising marker of fibrotic remodeling for the investigation, diagnosis, and therapeutic development of pulmonary fibrosis.

Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices.

Long-range axonal projections of diverse classes of neocortical excitatory neurons likely contribute to brain-wide interactions processing sensory, cognitive and motor signals. Here, we performed light-sheet imaging of fluorescently labeled axons from genetically defined neurons located in posterior primary somatosensory barrel cortex and supplemental somatosensory cortex. We used convolutional networks to segment axon-containing voxels and quantified their distribution within the Allen Mouse Brain Atlas Common Coordinate Framework. Axonal density was analyzed for different classes of glutamatergic neurons using transgenic mouse lines selectively expressing Cre recombinase in layer 2/3 intratelencephalic projection neurons (Rasgrf2-dCre), layer 4 intratelencephalic projection neurons (Scnn1a-Cre), layer 5 intratelencephalic projection neurons (Tlx3-Cre), layer 5 pyramidal tract projection neurons (Sim1-Cre), layer 5 projection neurons (Rbp4-Cre), and layer 6 corticothalamic neurons (Ntsr1-Cre). We found distinct axonal projections from the different neuronal classes to many downstream brain areas, which were largely similar for primary and supplementary somatosensory cortices. Functional connectivity maps obtained from optogenetic activation of sensory cortex and wide-field imaging revealed topographically organized evoked activity in frontal cortex with neurons located more laterally in somatosensory cortex signaling to more anteriorly located regions in motor cortex, consistent with the anatomical projections. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling.

Epilepsy insights revealed by intravital functional optical imaging.

Despite an abundance of pharmacologic and surgical epilepsy treatments, there remain millions of patients suffering from poorly controlled seizures. One approach to closing this treatment gap may be found through a deeper mechanistic understanding of the network alterations that underly this aberrant activity. Functional optical imaging in vertebrate models provides powerful advantages to this end, enabling the spatiotemporal acquisition of individual neuron activity patterns across multiple seizures. This coupled with the advent of genetically encoded indicators, be them for specific ions, neurotransmitters or voltage, grants researchers unparalleled access to the intact nervous system. Here, we will review how in vivo functional optical imaging in various vertebrate seizure models has advanced our knowledge of seizure dynamics, principally seizure initiation, propagation and termination.

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.

Decreased GABA levels during development result in increased connectivity in the larval zebrafish tectum.

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b, and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b-normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

ClearScope: a fully integrated light sheet theta microscope for sub-cellular resolution imaging without lateral size constraints.

Three-dimensional (3D) ex vivo imaging of cleared intact brains of animal models and large human and non-human primate postmortem brain specimens is important for understanding the physiological neural network connectivity patterns and the pathological alterations underlying neuropsychiatric and neurological disorders. Light-sheet microscopy has emerged as a highly effective imaging modality for rapid high-resolution imaging of large cleared samples. However, the orthogonal arrangements of illumination and detection optics in light sheet microscopy limits the size of specimen that can be imaged. Recently developed light sheet theta microscopy (LSTM) technology addressed this by utilizing a unique arrangement of two illumination light paths oblique to the detection light path, while allowing perpendicular arrangement of the detection light path relative to the specimen surface. Here, we report development of a next-generation, fully integrated, and user-friendly LSTM system for rapid sub-cellular resolution imaging uniformly throughout a large specimen without constraining the lateral (XY) size. In addition, we provide a seamlessly integrated workflow for image acquisition, data storage, pre- and post-processing, enhancement, and quantitative analysis. We demonstrate the system performance by high-resolution 3D imaging of intact mouse brains and human brain samples, and complete data analysis including digital neuron tracing, vessel reconstruction and design-based stereological analysis in 3D. This technically enhanced and user-friendly LSTM implementation will enable rapid quantitative mapping of molecular and cellular features of interests in diverse types of very large samples.

Deciphering the spatial distribution of Gli1-lineage cells in dental, oral, and craniofacial regions.

The craniofacial bone, crucial for protecting brain tissue and supporting facial structure, undergoes continuous remodeling through mesenchymal (MSCs) or skeletal stem cells (SSCs) in their niches. Gli1 is an ideal marker for labeling MSCs and osteoprogenitors in this region, and Gli1-lineage cells are identified as pivotal for bone growth, development, repair, and regeneration. Despite its significance, the distribution of Gli1-lineage cells across the dental, oral, and craniofacial (DOC) regions remains to be systematically explored. Utilizing tissue-clearing and light sheet fluorescence microscopy (LSFM) with a Gli1CreER; tdTomatoAi14 mouse model, we mapped the spatial distribution of Gli1-lineage cells throughout the skull, focusing on calvarial bones, sutures, bone marrow, teeth, periodontium, jaw bones, and the temporomandibular joint (TMJ). We found Gli1-lineage cells widespread in these areas, underscoring their significance in DOC regions. Additionally, we observed their role in repairing calvarial bone defects, providing novel insights into craniofacial biology and stem cell niches and enhancing our understanding of stem cells and their progeny's behavior in vivo.

This study investigates the presence and role of a specific stem cell population, known as Gli1-lineage cells, in various parts of the skull and facial bones. Using advanced imaging techniques, we found that these cells are widely distributed across the dental, oral, and craniofacial regions, especially in the cranial sutures, teeth, and jaw. Notably, Gli1-lineage cells migrate to the injury site, which is essential in bone repair and regeneration. These findings enhance our understanding of how stem cells contribute to healing and development in the craniofacial region.

Toxoplasma gondii Infection of Alzheimer's Disease Mice Reduces Brain Amyloid Density Globally and Regionally.

BACKGROUND: Toxoplasma gondii infection of Alzheimer's disease model mice decreases amyloid ß plaques. We aimed to determine if there is a brain regional difference in amyloid ß reduction in the brains of T. gondii-infected compared to control mice. METHOD: Three-month-old 5xFAD (AD model) mice were injected with T. gondii or with phosphate-buffered saline as a control. Intact brains were harvested at 6 weeks postinfection, optically cleared using iDISCO+, and brain-wide amyloid burden was visualized using volumetric light-sheet imaging. Amyloid signal was quantified across each brain and computationally mapped to the Allen Institute Brain Reference Atlas to determine amyloid density in each region. RESULTS: A brain-wide analysis of amyloid in control and T. gondii-infected 5xFAD mice revealed that T. gondii infection decreased amyloid burden in the brain globally as well as in the cortex and hippocampus, and many daughter regions. Daughter regions that showed reduced amyloid burden included the prelimbic cortex, visual cortex, and retrosplenial cortex. The olfactory tubercle, a region known to have increased monocytes following T. gondii infection, also showed reduced amyloid after infection. CONCLUSIONS: T. gondii infection of AD mice reduces amyloid burden in a brain region-specific manner that overlaps with known regions of T. gondii infection and peripheral immune cell infiltration.

Arterial specification precedes microvascular restitution in the peri-infarct cortex that is driven by small microvessels.

Evaluation of microvascular networks was impeded until recently by the need of histological tissue sectioning, which precluded 3D analyses. Using light-sheet microscopy, we investigated microvascular network characteristics in the peri-infarct cortex of mice 3-56 days after transient middle cerebral artery occlusion. In animal subgroups, the sphingosine-1-phosphate analog FTY720 (Fingolimod) was administered starting 24 hours post-ischemia. Light-sheet microscopy revealed a striking pattern of microvascular changes in the peri-infarct cortex, that is, a loss of microvessels, which was most prominent after 7 days and followed by the reappearance of microvessels over 56 days which revealed an increased branching point density and shortened branches. Using a novel AI-based image analysis algorithm we found that the length density of microvessels expressing the arterial specification marker α-smooth muscle actin markedly increased in the peri-infarct cortex already at 7 days post-ischemia. The length and branch density of small microvessels, but not of intermediate or large microvessels increased above pre-ischemic levels within 14-56 days. FTY720 increased the length and branch density of small microvessels. This study demonstrates long-term alterations of microvascular architecture post-ischemia indicative of increased collateralization most notably of small microvessels. Light-sheet microscopy will greatly advance the assessment of microvascular responses to restorative stroke therapies.

Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy.

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

Mirror-assisted light-sheet microscopy: a simple upgrade to enable bi-directional sample excitation.

Significance: Light-sheet microscopy is a powerful imaging technique that achieves optical sectioning via selective illumination of individual sample planes. However, when the sample contains opaque or scattering tissues, the incident light sheet may not be able to uniformly excite the entire sample. For example, in the context of larval zebrafish whole-brain imaging, occlusion by the eyes prevents access to a significant portion of the brain during common implementations using unidirectional illumination. Aim: We introduce mirror-assisted light-sheet microscopy (mLSM), a simple inexpensive method that can be implemented on existing digitally scanned light-sheet setups by adding a single optical element-a mirrored micro-prism. The prism is placed near the sample to generate a second excitation path for accessing previously obstructed regions. Approach: Scanning the laser beam onto the mirror generates a second, reflected illumination path that circumvents the occlusion. Online tuning of the scanning and laser power waveforms enables near uniform sample excitation with dual illumination. Results: mLSM produces cellular-resolution images of zebrafish brain regions inaccessible to unidirectional illumination. The imaging quality in regions illuminated by the direct or reflected sheet is adjustable by translating the excitation objective. The prism does not interfere with visually guided behavior. Conclusions: mLSM presents an easy-to-implement, cost-effective way to upgrade an existing light-sheet system to obtain more imaging data from a biological sample.

Scattering of a Bessel Pincer Light-Sheet Beam on a Charged Particle at Arbitrary Size.

Electromagnetic scattering is a routine tool for rapid, non-contact characterization of particle media. In previous work, the interaction targets of scattering intensity, scattering efficiency, and extinction efficiency of Bessel pincer light-sheet beams were all aimed at dielectric spheres. However, most particles in nature are charged. Considering the boundary condition on a charged sphere, the beam shape coefficients (BSCs) (pmn,qmn) of the charged spherical particle illuminated by a Bessel pincer light-sheet beam are obtained. The extinction, scattering, and absorption efficiencies are derived under the generalized Lorenz-Mie theory (GLMT) framework. This study reveals the significant differences in scattering characteristics of Bessel pincer light-sheet beams on a charged particle compared to traditional beams. The simulations show a few apparent differences in the far-field scattering intensity and efficiencies between charged and natural spheres under the influence of dimensionless size parameters. As dimensionless parameters increase, the difference between the charged and neutral spheres decreases. The effects of refractive index and beam parameters on scattering, extinction, and absorption coefficients are different but tend to converge with increasing dimensionless parameters. When applied to charged spheres with different refractive indices, the scattering, extinction, and absorption efficiencies of Bessel pincer light-sheet beams change with variations in surface charge. However, once the surface charge reaches saturation, these efficiencies become stable. This study is significant for understanding optical manipulation and super-resolution imaging in single-molecule microbiology.

Light-Sheet Microscopy Enables Three-Dimensional Fluorescence Imaging and Live Imaging of Satellite Cells on Skeletal Muscle Fibers.

The ex vivo myofiber culture system has proven to be a useful methodology to explore the biology and behavior of satellite cells within their niche environment. However, a limitation of this system is that myofibers and their associated satellite cells are commonly examined using conventional fluorescence microscopy, which renders a three-dimensional system into two-dimensional imaging, leading to the loss of precious information or misleading interpretation of observations. Here, we report on the use of light-sheet fluorescence microscopy to generate three-dimensional and live imaging of satellite cells on myofibers. Light-sheet microscopy offers high imaging speed and good spatial resolution with minimal photo-bleaching, allowing live imaging and three-dimensional acquisition of skeletal muscle fiber specimen. The potentials of this technology are wide, ranging from the visualization of satellite cell behavior such as cell division and cell migration to imaging the sub-cellular localization of proteins or organelles.

Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.

The central oxytocinergic system of the prairie vole.

Oxytocin (OXT) is a peptide hormone and a neuropeptide that regulates various peripheral physiological processes and modulates behavioral responses in the central nervous system. While the humoral release occurs from the axons arriving at the median eminence, the neuropeptide is also released from oxytocinergic cell axons in various brain structures that contain its receptor, and from their dendrites in hypothalamic nuclei and potentially into the cerebrospinal fluid (CSF). Understanding oxytocin's complex functions requires the knowledge on patterns of oxytocinergic projections in relationship to its receptor (OXTR). This study provides the first comprehensive examination of the oxytocinergic system in the prairie vole (Microtus ochrogaster), an animal exhibiting social behaviors that mirror human social behaviors linked to oxytocinergic functioning. Using light and electron microscopy, we characterized the neuroanatomy of the oxytocinergic system in this species. OXT+ cell bodies were found primarily in the hypothalamus, and axons were densest in subcortical regions. Examination of the OXT+ fibers and their relationship to oxytocin receptor transcripts (Oxtr) revealed that except for some subcortical structures, the presence of axons was not correlated with the amount of Oxtr across the brain. Of particular interest, the cerebral cortex that had high expression of Oxtr transcripts contained little to no fibers. Electron microscopy is used to quantify dense cored vesicles (DCV) in OXT+ axons and to identify potential axonal release sites. The ependymal cells that line the ventricles were frequently permissive of DCV-containing OXT+ dendrites reaching the third ventricle. Our results highlight a mechanism in which oxytocin is released directly into the ventricles and circulates throughout the ventricular system, may serve as the primary source for oxytocin that binds to OXTR in the cerebral cortex.