Post-transcriptional splicing can occur in a slow-moving zone around the gene.

Splicing is the stepwise molecular process by which introns are removed from pre-mRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing.

QuATON: Quantization Aware Training of Optical Neurons.

Optical processors, built with "optical neurons", can efficiently perform high-dimensional linear operations at the speed of light. Thus they are a promising avenue to accelerate large-scale linear computations. With the current advances in micro-fabrication, such optical processors can now be 3D fabricated, but with a limited precision. This limitation translates to quantization of learnable parameters in optical neurons, and should be handled during the design of the optical processor in order to avoid a model mismatch. Specifically, optical neurons should be trained or designed within the physical-constraints at a predefined quantized precision level. To address this critical issues we propose a physics-informed quantization-aware training framework. Our approach accounts for physical constraints during the training process, leading to robust designs. We demonstrate that our approach can design state of the art optical processors using diffractive networks for multiple physics based tasks despite quantized learnable parameters. We thus lay the foundation upon which improved optical processors may be 3D fabricated in the future.

Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues.

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

Multisensory gamma stimulation promotes glymphatic clearance of amyloid.

The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

Fast light-field 3D microscopy with out-of-distribution detection and adaptation through conditional normalizing flows.

Real-time 3D fluorescence microscopy is crucial for the spatiotemporal analysis of live organisms, such as neural activity monitoring. The eXtended field-of-view light field microscope (XLFM), also known as Fourier light field microscope, is a straightforward, single snapshot solution to achieve this. The XLFM acquires spatial-angular information in a single camera exposure. In a subsequent step, a 3D volume can be algorithmically reconstructed, making it exceptionally well-suited for real-time 3D acquisition and potential analysis. Unfortunately, traditional reconstruction methods (like deconvolution) require lengthy processing times (0.0220 Hz), hampering the speed advantages of the XLFM. Neural network architectures can overcome the speed constraints but do not automatically provide a way to certify the realism of their reconstructions, which is essential in the biomedical realm. To address these shortcomings, this work proposes a novel architecture to perform fast 3D reconstructions of live immobilized zebrafish neural activity based on a conditional normalizing flow. It reconstructs volumes at 8 Hz spanning 512x512x96 voxels, and it can be trained in under two hours due to the small dataset requirements (50 image-volume pairs). Furthermore, normalizing flows provides a way to compute the exact likelihood of a sample. This allows us to certify whether the predicted output is in- or ood, and retrain the system when a novel sample is detected. We evaluate the proposed method on a cross-validation approach involving multiple in-distribution samples (genetically identical zebrafish) and various out-of-distribution ones.

Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding.

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells at nanoscale resolution; however, current methods cannot be performed in multi-well cell culture plates which limits the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables consistent ~4.2x expansion within individual wells, across multiple wells, and between plates processed in parallel. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, in human cardiomyocytes (CMs) based on Hoechst signal intensity. We show a dose dependent effect on nuclear chromatin that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Temporally multiplexed imaging of dynamic signaling networks in living cells.

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Non-invasive temporal interference electrical stimulation of the human hippocampus.

Deep brain stimulation (DBS) via implanted electrodes is used worldwide to treat patients with severe neurological and psychiatric disorders. However, its invasiveness precludes widespread clinical use and deployment in research. Temporal interference (TI) is a strategy for non-invasive steerable DBS using multiple kHz-range electric fields with a difference frequency within the range of neural activity. Here we report the validation of the non-invasive DBS concept in humans. We used electric field modeling and measurements in a human cadaver to verify that the locus of the transcranial TI stimulation can be steerably focused in the hippocampus with minimal exposure to the overlying cortex. We then used functional magnetic resonance imaging and behavioral experiments to show that TI stimulation can focally modulate hippocampal activity and enhance the accuracy of episodic memories in healthy humans. Our results demonstrate targeted, non-invasive electrical stimulation of deep structures in the human brain.

Optogenetic control of neural activity: The biophysics of microbial rhodopsins in neuroscience.

Optogenetics, the use of microbial rhodopsins to make the electrical activity of targeted neurons controllable by light, has swept through neuroscience, enabling thousands of scientists to study how specific neuron types contribute to behaviors and pathologies, and how they might serve as novel therapeutic targets. By activating a set of neurons, one can probe what functions they can initiate or sustain, and by silencing a set of neurons, one can probe the functions they are necessary for. We here review the biophysics of these molecules, asking why they became so useful in neuroscience for the study of brain circuitry. We review the history of the field, including early thinking, early experiments, applications of optogenetics, pre-optogenetics targeted neural control tools, and the history of discovering and characterizing microbial rhodopsins. We then review the biophysical attributes of rhodopsins that make them so useful to neuroscience - their classes and structure, their photocycles, their photocurrent magnitudes and kinetics, their action spectra, and their ion selectivity. Our hope is to convey to the reader how specific biophysical properties of these molecules made them especially useful to neuroscientists for a difficult problem - the control of high-speed electrical activity, with great precision and ease, in the brain.

Expansion microscopy using a single anchor molecule for high-yield multiplexed imaging of proteins and RNAs.

Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to existing ExM protocols is the equipping of different kinds of molecules, with different kinds of anchoring moieties, so they can all be pulled apart from each other by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables proteins and RNAs to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and reduces cost (by 2-10-fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this united ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructures, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues and may support the visualization of other kinds of biomolecular species as well. uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Fast light-field 3D microscopy with out-of-distribution detection and adaptation through Conditional Normalizing Flows.

Real-time 3D fluorescence microscopy is crucial for the spatiotemporal analysis of live organisms, such as neural activity monitoring. The eXtended field-of-view light field microscope (XLFM), also known as Fourier light field microscope, is a straightforward, single snapshot solution to achieve this. The XLFM acquires spatial-angular information in a single camera exposure. In a subsequent step, a 3D volume can be algorithmically reconstructed, making it exceptionally well-suited for real-time 3D acquisition and potential analysis. Unfortunately, traditional reconstruction methods (like deconvolution) require lengthy processing times (0.0220 Hz), hampering the speed advantages of the XLFM. Neural network architectures can overcome the speed constraints at the expense of lacking certainty metrics, which renders them untrustworthy for the biomedical realm. This work proposes a novel architecture to perform fast 3D reconstructions of live immobilized zebrafish neural activity based on a conditional normalizing flow. It reconstructs volumes at 8 Hz spanning 512 × 512 × 96 voxels, and it can be trained in under two hours due to the small dataset requirements (10 image-volume pairs). Furthermore, normalizing flows allow for exact Likelihood computation, enabling distribution monitoring, followed by out-of-distribution detection and retraining of the system when a novel sample is detected. We evaluate the proposed method on a cross-validation approach involving multiple in-distribution samples (genetically identical zebrafish) and various out-of-distribution ones.

Vibrotactile stimulation at gamma frequency mitigates pathology related to neurodegeneration and improves motor function.

The risk for neurodegenerative diseases increases with aging, with various pathological conditions and functional deficits accompanying these diseases. We have previously demonstrated that non-invasive visual stimulation using 40 Hz light flicker ameliorated pathology and modified cognitive function in mouse models of neurodegeneration, but whether 40 Hz stimulation using another sensory modality can impact neurodegeneration and motor function has not been studied. Here, we show that whole-body vibrotactile stimulation at 40 Hz leads to increased neural activity in the primary somatosensory cortex (SSp) and primary motor cortex (MOp). In two different mouse models of neurodegeneration, Tau P301S and CK-p25 mice, daily exposure to 40 Hz vibrotactile stimulation across multiple weeks also led to decreased brain pathology in SSp and MOp. Furthermore, both Tau P301S and CK-p25 mice showed improved motor performance after multiple weeks of daily 40 Hz vibrotactile stimulation. Vibrotactile stimulation at 40 Hz may thus be considered as a promising therapeutic strategy for neurodegenerative diseases with motor deficits.

Cell-type specific defects in PTEN-mutant cortical organoids converge on abnormal circuit activity.

De novo heterozygous loss-of-function mutations in phosphatase and tensin homolog (PTEN) are strongly associated with autism spectrum disorders; however, it is unclear how heterozygous mutations in this gene affect different cell types during human brain development and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics and spatial transcriptomics and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep-layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.

3D Domain Adaptive Instance Segmentation via Cyclic Segmentation GANs.

3D instance segmentation for unlabeled imaging modalities is a challenging but essential task as collecting expert annotation can be expensive and time-consuming. Existing works segment a new modality by either deploying pre-trained models optimized on diverse training data or sequentially conducting image translation and segmentation with two relatively independent networks. In this work, we propose a novel Cyclic Segmentation Generative Adversarial Network (CySGAN) that conducts image translation and instance segmentation simultaneously using a unified network with weight sharing. Since the image translation layer can be removed at inference time, our proposed model does not introduce additional computational cost upon a standard segmentation model. For optimizing CySGAN, besides the CycleGAN losses for image translation and supervised losses for the annotated source domain, we also utilize self-supervised and segmentation-based adversarial objectives to enhance the model performance by leveraging unlabeled target domain images. We benchmark our approach on the task of 3D neuronal nuclei segmentation with annotated electron microscopy (EM) images and unlabeled expansion microscopy (ExM) data. The proposed CySGAN outperforms pre-trained generalist models, feature-level domain adaptation models, and the baselines that conduct image translation and segmentation sequentially. Our implementation and the newly collected, densely annotated ExM zebrafish brain nuclei dataset, named NucExM, are publicly available at https://connectomics-bazaar.github.io/proj/CySGAN/index.html.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

Dynamic Changes in Heparan Sulfate Nanostructure in Human Pluripotent Stem Cell Differentiation.

Heparan sulfate (HS) is a heterogeneous, cell-surface polysaccharide critical for transducing signals essential for mammalian development. Imaging of signaling proteins has revealed how their localization influences their information transfer. In contrast, the contribution of the spatial distribution and nanostructure of information-rich, signaling polysaccharides like HS is not known. Using expansion microscopy (ExM), we found striking changes in HS nanostructure occur as human pluripotent stem (hPS) cells differentiate, and these changes correlate with growth factor signaling. Our imaging studies show that undifferentiated hPS cells are densely coated with HS displayed as hair-like protrusions. This ultrastructure can recruit fibroblast growth factor for signaling. When the hPS cells differentiate into the ectoderm lineage, HS is localized into dispersed puncta. This striking change in HS distribution coincides with a decrease in fibroblast growth factor binding to neural cells. While developmental variations in HS sequence were thought to be the primary driver of alterations in HS-mediated growth factor signaling, our high-resolution images indicate a role for the HS nanostructure. Our study highlights the utility of high-resolution glycan imaging using ExM. In the case of HS, we found that changes in how the polysaccharide is displayed link to profound differences in growth factor binding.

Recording of cellular physiological histories along optically readable self-assembling protein chains.

Observing cellular physiological histories is key to understanding normal and disease-related processes. Here we describe expression recording islands-a fully genetically encoded approach that enables both continual digital recording of biological information within cells and subsequent high-throughput readout in fixed cells. The information is stored in growing intracellular protein chains made of self-assembling subunits, human-designed filament-forming proteins bearing different epitope tags that each correspond to a different cellular state or function (for example, gene expression downstream of neural activity or pharmacological exposure), allowing the physiological history to be read out along the ordered subunits of protein chains with conventional optical microscopy. We use expression recording islands to record gene expression timecourse downstream of specific pharmacological and physiological stimuli in cultured neurons and in living mouse brain, with a time resolution of a fraction of a day, over periods of days to weeks.