107 results on '"Shroff, Hari"'
Search Results
2. Convolutional neural network transformer (CNNT) for fluorescence microscopy image denoising with improved generalization and fast adaptation.
- Author
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Rehman A, Zhovmer A, Sato R, Mukouyama YS, Chen J, Rissone A, Puertollano R, Liu J, Vishwasrao HD, Shroff H, Combs CA, and Xue H
- Abstract
Deep neural networks can improve the quality of fluorescence microscopy images. Previous methods, based on Convolutional Neural Networks (CNNs), require time-consuming training of individual models for each experiment, impairing their applicability and generalization. In this study, we propose a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), that outperforms CNN based networks for image denoising. We train a general CNNT based backbone model from pairwise high-low Signal-to-Noise Ratio (SNR) image volumes, gathered from a single type of fluorescence microscope, an instant Structured Illumination Microscope. Fast adaptation to new microscopes is achieved by fine-tuning the backbone on only 5-10 image volume pairs per new experiment. Results show that the CNNT backbone and fine-tuning scheme significantly reduces training time and improves image quality, outperforming models trained using only CNNs such as 3D-RCAN and Noise2Fast. We show three examples of efficacy of this approach in wide-field, two-photon, and confocal fluorescence microscopy., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
- Published
- 2024
- Full Text
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3. Deep learning-based aberration compensation improves contrast and resolution in fluorescence microscopy.
- Author
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Guo M, Wu Y, Hobson CM, Su Y, Qian S, Krueger E, Christensen R, Kroeschell G, Bui J, Chaw M, Zhang L, Liu J, Hou X, Han X, Lu Z, Ma X, Zhovmer A, Combs C, Moyle M, Yemini E, Liu H, Liu Z, Benedetto A, La Riviere P, Colón-Ramos D, and Shroff H
- Abstract
Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics into the imaging path. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations and experiments to show that applying the trained 'de-aberration' networks outperforms alternative methods, providing restoration on par with adaptive optics techniques; and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in C. elegans embryos.
- Published
- 2024
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4. Live-cell imaging powered by computation.
- Author
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Shroff H, Testa I, Jug F, and Manley S
- Subjects
- Humans, Animals, Image Processing, Computer-Assisted methods, Artificial Intelligence, Signal-To-Noise Ratio, Cell Survival, Microscopy, Fluorescence methods
- Abstract
The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Computational methods can help to address this challenge and are now shifting the boundaries of what is possible to capture in living systems. In this Review, we discuss these computational methods focusing on artificial intelligence-based approaches that can be layered on top of commonly used existing microscopies as well as hybrid methods that integrate computation and microscope hardware. We specifically discuss how computational approaches can improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging., (© 2024. Springer Nature Limited.)
- Published
- 2024
- Full Text
- View/download PDF
5. Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.
- Author
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Miyoshi T, Vishwasrao H, Belyantseva I, Sajeevadathan M, Ishibashi Y, Adadey S, Harada N, Shroff H, and Friedman T
- Abstract
Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles., Competing Interests: Declaration of interests The authors declare no conflict of interest associated with this manuscript.
- Published
- 2024
- Full Text
- View/download PDF
6. Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore.
- Author
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Fiore A, Yu G, Northey JJ, Patel R, Ravenscroft TA, Ikegami R, Kolkman W, Kumar P, Grimm JB, Dilan TL, Ruetten VMS, Ahrens MB, Shroff H, Lavis LD, Wang S, Weaver VM, and Pedram K
- Abstract
All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology., Competing Interests: Competing Interests A patent application relating to this work has been filed by the Howard Hughes Medical Institute (internal reference 2024-017-01).
- Published
- 2024
- Full Text
- View/download PDF
7. Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.
- Author
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Miyoshi T, Vishwasrao HD, Belyantseva IA, Sajeevadathan M, Ishibashi Y, Adadey SM, Harada N, Shroff H, and Friedman TB
- Abstract
Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles., Competing Interests: Declaration of interests The authors declare no conflict of interest associated with this manuscript.
- Published
- 2024
- Full Text
- View/download PDF
8. Convolutional Neural Network Transformer (CNNT) for Fluorescence Microscopy image Denoising with Improved Generalization and Fast Adaptation.
- Author
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Rehman A, Zhovmer A, Sato R, Mukoyama Y, Chen J, Rissone A, Puertollano R, Vishwasrao H, Shroff H, Combs CA, and Xue H
- Abstract
Deep neural networks have been applied to improve the image quality of fluorescence microscopy imaging. Previous methods are based on convolutional neural networks (CNNs) which generally require more time-consuming training of separate models for each new imaging experiment, impairing the applicability and generalization. Once the model is trained (typically with tens to hundreds of image pairs) it can then be used to enhance new images that are like the training data. In this study, we proposed a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), to outperform the CNN networks for image denoising. In our scheme we have trained a single CNNT based "backbone model" from pairwise high-low SNR images for one type of fluorescence microscope (instance structured illumination, iSim). Fast adaption to new applications was achieved by fine-tuning the backbone on only 5-10 sample pairs per new experiment. Results show the CNNT backbone and fine-tuning scheme significantly reduces the training time and improves the image quality, outperformed training separate models using CNN approaches such as - RCAN and Noise2Fast. Here we show three examples of the efficacy of this approach on denoising wide-field, two-photon and confocal fluorescence data. In the confocal experiment, which is a 5×5 tiled acquisition, the fine-tuned CNNT model reduces the scan time form one hour to eight minutes, with improved quality., Competing Interests: Conflict of Interest The authors declare no conflict of interest.
- Published
- 2024
9. Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.
- Author
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Updegrove TB, Delerue T, Anantharaman V, Cho H, Chan C, Nipper T, Choo-Wosoba H, Jenkins LM, Zhang L, Su Y, Shroff H, Chen J, Bewley CA, Aravind L, and Ramamurthi KS
- Abstract
Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness., Competing Interests: Competing Interests: The authors declare no competing interests.
- Published
- 2024
- Full Text
- View/download PDF
10. Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis .
- Author
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Chareyre S, Li X, Anjuwon-Foster BR, Updegrove TB, Clifford S, Brogan AP, Su Y, Zhang L, Chen J, Shroff H, and Ramamurthi KS
- Subjects
- Transcriptional Activation, Spores, Bacterial genetics, Spores, Bacterial metabolism, Cell Division genetics, Sigma Factor genetics, Sigma Factor metabolism, Bacillus subtilis metabolism, Bacterial Proteins metabolism
- Abstract
When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σ
F is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program., Competing Interests: Competing interests statement:The authors declare no competing interest.- Published
- 2024
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- View/download PDF
11. Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM).
- Author
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Chandler T, Guo M, Su Y, Chen J, Wu Y, Liu J, Agashe A, Fischer RS, Mehta SB, Kumar A, Baskin TI, Jamouillé V, Liu H, Swaminathan V, Nain A, Oldenbourg R, Riviére P, and Shroff H
- Abstract
Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales., Competing Interests: Conflict of interest/Competing interests. H. S., A. K., S. M., P. L. R., R. O., Y. W., and T. C. hold US Patent # 11428632. Ethics approval and consent to participate. Not applicable.
- Published
- 2024
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12. Phase diversity-based wavefront sensing for fluorescence microscopy.
- Author
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Johnson C, Guo M, Schneider MC, Su Y, Khuon S, Reiser N, Wu Y, La Riviere P, and Shroff H
- Abstract
Fluorescence microscopy is an invaluable tool in biology, yet its performance is compromised when the wavefront of light is distorted due to optical imperfections or the refractile nature of the sample. Such optical aberrations can dramatically lower the information content of images by degrading image contrast, resolution, and signal. Adaptive optics (AO) methods can sense and subsequently cancel the aberrated wavefront, but are too complex, inefficient, slow, or expensive for routine adoption by most labs. Here we introduce a rapid, sensitive, and robust wavefront sensing scheme based on phase diversity, a method successfully deployed in astronomy but underused in microscopy. Our method enables accurate wavefront sensing to less than λ/35 root mean square (RMS) error with few measurements, and AO with no additional hardware besides a corrective element. After validating the method with simulations, we demonstrate calibration of a deformable mirror > 100-fold faster than comparable methods (corresponding to wavefront sensing on the ~100 ms scale), and sensing and subsequent correction of severe aberrations (RMS wavefront distortion exceeding λ/2), restoring diffraction-limited imaging on extended biological samples., Competing Interests: Competing Interests C.J., M.G., P.J.L., and H.S. have filed a provisional patent for the technique described here.
- Published
- 2024
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13. Author Correction: Experimental and theoretical model for the origin of coiling of cellular protrusions around fibers.
- Author
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Sadhu RK, Hernandez-Padilla C, Eisenbach YE, Penič S, Zhang L, Vishwasrao HD, Behkam B, Konstantopoulos K, Shroff H, Iglič A, Peles E, Nain AS, and Gov NS
- Published
- 2023
- Full Text
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14. Experimental and theoretical model for the origin of coiling of cellular protrusions around fibers.
- Author
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Sadhu RK, Hernandez-Padilla C, Eisenbach YE, Penič S, Zhang L, Vishwasrao HD, Behkam B, Konstantopoulos K, Shroff H, Iglič A, Peles E, Nain AS, and Gov NS
- Subjects
- Endocytosis, Membrane Proteins, Models, Theoretical, Cell Surface Extensions, Cues
- Abstract
Protrusions at the leading-edge of a cell play an important role in sensing the extracellular cues during cellular spreading and motility. Recent studies provided indications that these protrusions wrap (coil) around the extracellular fibers. However, the physics of this coiling process, and the mechanisms that drive it, are not well understood. We present a combined theoretical and experimental study of the coiling of cellular protrusions on fibers of different geometry. Our theoretical model describes membrane protrusions that are produced by curved membrane proteins that recruit the protrusive forces of actin polymerization, and identifies the role of bending and adhesion energies in orienting the leading-edges of the protrusions along the azimuthal (coiling) direction. Our model predicts that the cell's leading-edge coils on fibers with circular cross-section (above some critical radius), but the coiling ceases for flattened fibers of highly elliptical cross-section. These predictions are verified by 3D visualization and quantitation of coiling on suspended fibers using Dual-View light-sheet microscopy (diSPIM). Overall, we provide a theoretical framework, supported by experiments, which explains the physical origin of the coiling phenomenon., (© 2023. Springer Nature Limited.)
- Published
- 2023
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15. Three-dimensional structured illumination microscopy with enhanced axial resolution.
- Author
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Li X, Wu Y, Su Y, Rey-Suarez I, Matthaeus C, Updegrove TB, Wei Z, Zhang L, Sasaki H, Li Y, Guo M, Giannini JP, Vishwasrao HD, Chen J, Lee SJ, Shao L, Liu H, Ramamurthi KS, Taraska JW, Upadhyaya A, La Riviere P, and Shroff H
- Subjects
- Microscopy, Fluorescence methods, Cytoskeleton, Lysosomes, Lighting, Imaging, Three-Dimensional methods
- Abstract
The axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
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16. Cell division machinery drives cell-specific gene activation during bacterial differentiation.
- Author
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Chareyre S, Li X, Anjuwon-Foster BR, Clifford S, Brogan A, Su Y, Shroff H, and Ramamurthi KS
- Abstract
When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σ
F is exclusively activated in the smaller daughter cell. Compartment specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that a unique feature of the sporulation septum, defined by the cell division machinery, drives the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program., Competing Interests: The authors declare no competing interests.- Published
- 2023
- Full Text
- View/download PDF
17. Phase Diverse Phase Retrieval for Microscopy: Comparison of Gaussian and Poisson Approaches.
- Author
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Reiser N, Guo M, Shroff H, and Riviere PJ
- Abstract
Phase diversity is a widefield aberration correction method that uses multiple images to estimate the phase aberration at the pupil plane of an imaging system by solving an optimization problem. This estimated aberration can then be used to deconvolve the aberrated image or to reacquire it with aberration corrections applied to a deformable mirror. The optimization problem for aberration estimation has been formulated for both Gaussian and Poisson noise models but the Poisson model has never been studied in microscopy nor compared with the Gaussian model. Here, the Gaussian- and Poisson-based estimation algorithms are implemented and compared for widefield microscopy in simulation. The Poisson algorithm is found to match or outperform the Gaussian algorithm in a variety of situations, and converges in a similar or decreased amount of time. The Gaussian algorithm does perform better in low-light regimes when image noise is dominated by additive Gaussian noise. The Poisson algorithm is also found to be more robust to the effects of spatially variant aberration and phase noise. Finally, the relative advantages of re-acquisition with aberration correction and deconvolution with aberrated point spread functions are compared.
- Published
- 2023
18. Label-free cleared tissue microscopy and machine learning for 3D histopathology of biomaterial implants.
- Author
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Ngo TB, DeStefano S, Liu J, Su Y, Shroff H, Vishwasrao HD, and Sadtler K
- Subjects
- Extracellular Matrix, Machine Learning, Imaging, Three-Dimensional methods, Microscopy methods, Biocompatible Materials
- Abstract
Tissue clearing of whole intact organs has enhanced imaging by enabling the exploration of tissue structure at a subcellular level in three-dimensional space. Although clearing and imaging of the whole organ have been used to study tissue biology, the microenvironment in which cells evolve to adapt to biomaterial implants or allografts in the body is poorly understood. Obtaining high-resolution information from complex cell-biomaterial interactions with volumetric landscapes represents a key challenge in the fields of biomaterials and regenerative medicine. To provide a new approach to examine how tissue responds to biomaterial implants, we apply cleared tissue light-sheet microscopy and three-dimensional reconstruction to utilize the wealth of autofluorescence information for visualizing and contrasting anatomical structures. This study demonstrates the adaptability of the clearing and imaging technique to provide sub-cellular resolution (0.6 μm isotropic) 3D maps of various tissue types, using samples from fully intact peritoneal organs to volumetric muscle loss injury specimens. Specifically, in the volumetric muscle loss injury model, we provide 3D visualization of the implanted extracellular matrix biomaterial in the wound bed of the quadricep muscle groups and further apply computational-driven image classification to analyze the autofluorescence spectrum at multiple emission wavelengths to categorize tissue types at the injured site interacting with the biomaterial scaffolds., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)
- Published
- 2023
- Full Text
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19. Practical considerations for quantitative light sheet fluorescence microscopy.
- Author
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Hobson CM, Guo M, Vishwasrao HD, Wu Y, Shroff H, and Chew TL
- Subjects
- Microscopy, Fluorescence methods
- Abstract
Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses., (© 2022. Springer Nature America, Inc.)
- Published
- 2022
- Full Text
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20. An Exact Hypergraph Matching algorithm for posture identification in embryonic C. elegans.
- Author
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Lauziere A, Christensen R, Shroff H, and Balan R
- Subjects
- Animals, Algorithms, Cell Tracking, Heuristics, Caenorhabditis elegans, Posture
- Abstract
The nematode Caenorhabditis elegans (C. elegans) is a model organism used frequently in developmental biology and neurobiology [White, (1986), Sulston, (1983), Chisholm, (2016) and Rapti, (2020)]. The C. elegans embryo can be used for cell tracking studies to understand how cell movement drives the development of specific embryonic tissues. Analyses in late-stage development are complicated by bouts of rapid twitching motions which invalidate traditional cell tracking approaches. However, the embryo possesses a small set of cells which may be identified, thereby defining the coiled embryo's posture [Christensen, 2015]. The posture serves as a frame of reference, facilitating cell tracking even in the presence of twitching. Posture identification is nevertheless challenging due to the complete repositioning of the embryo between sampled images. Current approaches to posture identification rely on time-consuming manual efforts by trained users which limits the efficiency of subsequent cell tracking. Here, we cast posture identification as a point-set matching task in which coordinates of seam cell nuclei are identified to jointly recover the posture. Most point-set matching methods comprise coherent point transformations that use low order objective functions [Zhou, (2016) and Zhang, (2019)]. Hypergraphs, an extension of traditional graphs, allow more intricate modeling of relationships between objects, yet existing hypergraphical point-set matching methods are limited to heuristic algorithms which do not easily scale to handle higher degree hypergraphs [Duchenne, (2010), Chertok, (2010) and Lee, (2011)]. Our algorithm, Exact Hypergraph Matching (EHGM), adapts the classical branch-and-bound paradigm to dynamically identify a globally optimal correspondence between point-sets under an arbitrarily intricate hypergraphical model. EHGM with hypergraphical models inspired by C. elegans embryo shape identified posture more accurately (56%) than established point-set matching methods (27%), correctly identifying twice as many sampled postures as a leading graphical approach. Posterior region seeding empowered EHGM to correctly identify 78% of postures while reducing runtime, demonstrating the efficacy of the method on a cutting-edge problem in developmental biology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)
- Published
- 2022
- Full Text
- View/download PDF
21. Incorporating the image formation process into deep learning improves network performance.
- Author
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Li Y, Su Y, Guo M, Han X, Liu J, Vishwasrao HD, Li X, Christensen R, Sengupta T, Moyle MW, Rey-Suarez I, Chen J, Upadhyaya A, Usdin TB, Colón-Ramos DA, Liu H, Wu Y, and Shroff H
- Subjects
- Artifacts, Microscopy, Fluorescence, Image Processing, Computer-Assisted methods, Algorithms, Deep Learning
- Abstract
We present Richardson-Lucy network (RLN), a fast and lightweight deep learning method for three-dimensional fluorescence microscopy deconvolution. RLN combines the traditional Richardson-Lucy iteration with a fully convolutional network structure, establishing a connection to the image formation process and thereby improving network performance. Containing only roughly 16,000 parameters, RLN enables four- to 50-fold faster processing than purely data-driven networks with many more parameters. By visual and quantitative analysis, we show that RLN provides better deconvolution, better generalizability and fewer artifacts than other networks, especially along the axial dimension. RLN outperforms classic Richardson-Lucy deconvolution on volumes contaminated with severe out of focus fluorescence or noise and provides four- to sixfold faster reconstructions of large, cleared-tissue datasets than classic multi-view pipelines. We demonstrate RLN's performance on cells, tissues and embryos imaged with widefield-, light-sheet-, confocal- and super-resolution microscopy., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
- Published
- 2022
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22. Multiscale fluorescence imaging of living samples.
- Author
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Wu Y and Shroff H
- Subjects
- Microscopy, Fluorescence methods, Optical Imaging
- Abstract
Fluorescence microscopy is a highly effective tool for interrogating biological structure and function, particularly when imaging across multiple spatiotemporal scales. Here we survey recent innovations and applications in the relatively understudied area of multiscale fluorescence imaging of living samples. We discuss fundamental challenges in live multiscale imaging and describe successful examples that highlight the power of this approach. We attempt to synthesize general strategies from these test cases, aiming to help accelerate progress in this exciting area., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
- Published
- 2022
- Full Text
- View/download PDF
23. Nanotopography modulates cytoskeletal organization and dynamics during T cell activation.
- Author
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Wheatley BA, Rey-Suarez I, Hourwitz MJ, Kerr S, Shroff H, Fourkas JT, and Upadhyaya A
- Subjects
- Antigen-Presenting Cells metabolism, Cytoskeleton metabolism, T-Lymphocytes metabolism, Actins metabolism, Lymphocyte Activation
- Abstract
Exposure to MHC-antigen complexes on the surface of antigen-presenting cells (APCs) activates T cells, inducing the formation of the immune synapse (IS). Antigen detection at the APC surface is thus a critical step in the adaptive immune response. The physical properties of antigen-presenting surfaces encountered by T cells in vivo are believed to modulate T cell activation and proliferation. Although stiffness and ligand mobility influence IS formation, the effect of the complex topography of the APC surface on this process is not well understood. Here we investigate how nanotopography modulates cytoskeletal dynamics and signaling during the early stages of T cell activation using high-resolution fluorescence microscopy on nanofabricated surfaces with parallel nanoridges of different spacings. We find that although nanoridges reduce the maximum spread area as compared with cells on flat surfaces, the ridges enhance the accumulation of actin and the signaling kinase ZAP-70 at the IS. Actin polymerization is more dynamic in the presence of ridges, which influence the directionality of both actin flows and microtubule (MT) growth. Our results demonstrate that the topography of the activating surface exerts both global effects on T cell morphology and local changes in actin and MT dynamics, collectively influencing T cell signaling.
- Published
- 2022
- Full Text
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24. Stereotyped behavioral maturation and rhythmic quiescence in C. elegans embryos.
- Author
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Ardiel EL, Lauziere A, Xu S, Harvey BJ, Christensen RP, Nurrish S, Kaplan JM, and Shroff H
- Subjects
- Animals, Behavior, Animal physiology, Neurons physiology, Posture, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins physiology
- Abstract
Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here, we apply this approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were built using a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom tracking software. Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping nor the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in diverse animals and may play an important role in promoting normal developmental outcomes., Competing Interests: EA, AL, SX, BH, RC, SN, JK, HS No competing interests declared
- Published
- 2022
- Full Text
- View/download PDF
25. Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.
- Author
-
Zhao H, Nguyen A, Wu D, Li Y, Hassan SA, Chen J, Shroff H, Piszczek G, and Schuck P
- Abstract
Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by three to four different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced coassembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity., (Published by Oxford University Press on behalf of the National Academy of Sciences 2021.)
- Published
- 2022
- Full Text
- View/download PDF
26. The HIV-1 Viral Protease Is Activated during Assembly and Budding Prior to Particle Release.
- Author
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Tabler CO, Wegman SJ, Chen J, Shroff H, Alhusaini N, and Tilton JC
- Subjects
- Enzyme Activation physiology, HIV Infections virology, Humans, Protease Inhibitors pharmacology, HIV Protease metabolism, HIV-1 drug effects, HIV-1 enzymology
- Abstract
HIV-1 encodes a viral protease that is essential for the maturation of infectious viral particles. While protease inhibitors are effective antiretroviral agents, recent studies have shown that prematurely activating, rather than inhibiting, protease function leads to the pyroptotic death of infected cells, with exciting implications for efforts to eradicate viral reservoirs. Despite 40 years of research into the kinetics of protease activation, it remains unclear exactly when protease becomes activated. Recent reports have estimated that protease activation occurs minutes to hours after viral release, suggesting that premature protease activation is challenging to induce efficiently. Here, monitoring viral protease activity with sensitive techniques, including nanoscale flow cytometry and instant structured illumination microscopy, we demonstrate that the viral protease is activated within cells prior to the release of free virions. Using genetic mutants that lock protease into a precursor conformation, we further show that both the precursor and mature protease have rapid activation kinetics and that the activity of the precursor protease is sufficient for viral fusion with target cells. Our finding that HIV-1 protease is activated within producer cells prior to release of free virions helps resolve a long-standing question of when protease is activated and suggests that only a modest acceleration of protease activation kinetics is required to induce potent and specific elimination of HIV-infected cells. IMPORTANCE HIV-1 protease inhibitors have been a mainstay of antiretroviral therapy for more than 2 decades. Although antiretroviral therapy is effective at controlling HIV-1 replication, persistent reservoirs of latently infected cells quickly reestablish replication if therapy is halted. A promising new strategy to eradicate the latent reservoir involves prematurely activating the viral protease, which leads to the pyroptotic killing of infected cells. Here, we use highly sensitive techniques to examine the kinetics of protease activation during and shortly after particle formation. We found that protease is fully activated before virus is released from the cell membrane, which is hours earlier than recent estimates. Our findings help resolve a long-standing debate as to when the viral protease is initially activated during viral assembly and confirm that prematurely activating HIV-1 protease is a viable strategy to eradicate infected cells following latency reversal.
- Published
- 2022
- Full Text
- View/download PDF
27. Human deafness-associated variants alter the dynamics of key molecules in hair cell stereocilia F-actin cores.
- Author
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Miyoshi T, Belyantseva IA, Kitajiri SI, Miyajima H, Nishio SY, Usami SI, Kim BJ, Choi BY, Omori K, Shroff H, and Friedman TB
- Subjects
- Actins genetics, Animals, Formins, Hair metabolism, Humans, Mechanotransduction, Cellular genetics, Mice, Microfilament Proteins genetics, Stereocilia metabolism, Zebrafish genetics, Zebrafish metabolism, Deafness genetics, Deafness metabolism, Hearing Loss, Sensorineural metabolism
- Abstract
Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)
- Published
- 2022
- Full Text
- View/download PDF
28. Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche.
- Author
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Patel A, Wu Y, Han X, Su Y, Maugel T, Shroff H, and Roy S
- Subjects
- Animals, Drosophila melanogaster metabolism, Fibroblast Growth Factors metabolism, Muscles metabolism, Drosophila metabolism, Drosophila Proteins genetics, Drosophila Proteins metabolism
- Abstract
Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
29. Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein.
- Author
-
Zhao H, Nguyen A, Wu D, Li Y, Hassan SA, Chen J, Shroff H, Piszczek G, and Schuck P
- Abstract
Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.
- Published
- 2022
- Full Text
- View/download PDF
30. Multiview confocal super-resolution microscopy.
- Author
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Wu Y, Han X, Su Y, Glidewell M, Daniels JS, Liu J, Sengupta T, Rey-Suarez I, Fischer R, Patel A, Combs C, Sun J, Wu X, Christensen R, Smith C, Bao L, Sun Y, Duncan LH, Chen J, Pommier Y, Shi YB, Murphy E, Roy S, Upadhyaya A, Colón-Ramos D, La Riviere P, and Shroff H
- Subjects
- Animals, Caenorhabditis elegans cytology, Caenorhabditis elegans embryology, Caenorhabditis elegans growth & development, Cell Line, Tumor, Drosophila melanogaster cytology, Drosophila melanogaster growth & development, Humans, Imaginal Discs cytology, Mice, Myoblasts cytology, Organ Specificity, Single-Cell Analysis, Tissue Fixation, Deep Learning, Microscopy, Confocal methods, Microscopy, Confocal standards
- Abstract
Confocal microscopy
1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2 . Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)- Published
- 2021
- Full Text
- View/download PDF
31. Differential adhesion regulates neurite placement via a retrograde zippering mechanism.
- Author
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Sengupta T, Koonce NL, Vázquez-Martínez N, Moyle MW, Duncan LH, Emerson SE, Han X, Shao L, Wu Y, Santella A, Fan L, Bao Z, Mohler WA, Shroff H, and Colón-Ramos DA
- Subjects
- Animals, Caenorhabditis elegans Proteins metabolism, Cell Adhesion physiology, Gene Expression Regulation, Neurons physiology, Synapses, Brain cytology, Brain physiology, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, Cell Adhesion genetics, Neurites physiology
- Abstract
During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil , and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles., Competing Interests: TS, NK, NV, MM, LD, SE, XH, LS, YW, AS, LF, ZB, WM, HS, DC No competing interests declared
- Published
- 2021
- Full Text
- View/download PDF
32. Actomyosin dynamics modulate microtubule deformation and growth during T-cell activation.
- Author
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Rey-Suarez I, Rogers N, Kerr S, Shroff H, and Upadhyaya A
- Subjects
- Actin Cytoskeleton metabolism, Actin-Related Protein 2-3 Complex antagonists & inhibitors, Actin-Related Protein 2-3 Complex metabolism, Formins antagonists & inhibitors, Formins metabolism, Humans, Integrins metabolism, Jurkat Cells, Lymphocyte Activation, Microscopy, Fluorescence, Synapses immunology, T-Lymphocytes immunology, Thiones pharmacology, Uracil analogs & derivatives, Uracil pharmacology, Versicans metabolism, Actomyosin metabolism, Microtubules metabolism, T-Lymphocytes physiology
- Abstract
Activation of T-cells leads to the formation of immune synapses (ISs) with antigen-presenting cells. This requires T-cell polarization and coordination between the actomyosin and microtubule cytoskeletons. The interactions between these two cytoskeletal components during T-cell activation are not well understood. Here, we elucidate the interactions between microtubules and actin at the IS with high-resolution fluorescence microscopy. We show that microtubule growth dynamics in the peripheral actin-rich region is distinct from that in the central actin-free region. We further demonstrate that these differences arise from differential involvement of Arp2/3- and formin-nucleated actin structures. Formin inhibition results in a moderate decrease in microtubule growth rates, which is amplified in the presence of integrin engagement. In contrast, Arp2/3 inhibition leads to an increase in microtubule growth rates. We find that microtubule filaments are more deformed and exhibit greater shape fluctuations in the periphery of the IS than at the center. Using small molecule inhibitors, we show that actin dynamics and actomyosin contractility play key roles in defining microtubule deformations and shape fluctuations. Our results indicate a mechanical coupling between the actomyosin and microtubule systems during T-cell activation, whereby different actin structures influence microtubule dynamics in distinct ways.
- Published
- 2021
- Full Text
- View/download PDF
33. Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila .
- Author
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Elliott AD, Berndt A, Houpert M, Roy S, Scott RL, Chow CC, Shroff H, and White BH
- Subjects
- Animals, Behavior, Animal, Brain physiology, Computational Biology, Drosophila melanogaster physiology, Invertebrate Hormones physiology, Larva physiology, Molting, Motor Neurons, Receptors, Peptide, Drosophila physiology, Muscles anatomy & histology, Muscles physiology, Pupa physiology
- Abstract
Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila . Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution., Competing Interests: AE, AB, MH, SR, RS, CC, HS, BW No competing interests declared
- Published
- 2021
- Full Text
- View/download PDF
34. Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes.
- Author
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Chen J, Sasaki H, Lai H, Su Y, Liu J, Wu Y, Zhovmer A, Combs CA, Rey-Suarez I, Chang HY, Huang CC, Li X, Guo M, Nizambad S, Upadhyaya A, Lee SJ, Lucas LAG, and Shroff H
- Subjects
- Algorithms, Deep Learning, Image Processing, Computer-Assisted, Microscopy, Fluorescence methods
- Abstract
We demonstrate residual channel attention networks (RCAN) for the restoration and enhancement of volumetric time-lapse (four-dimensional) fluorescence microscopy data. First we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy four-dimensional super-resolution data, enabling image capture of over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables resolution enhancement equivalent to, or better than, other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy data as ground truth, achieving improvements of ~1.9-fold laterally and ~3.6-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluation and further enhancement of network performance.
- Published
- 2021
- Full Text
- View/download PDF
35. Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10.
- Author
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Campanello L, Traver MK, Shroff H, Schaefer BC, and Losert W
- Subjects
- Algorithms, Animals, Autophagosomes immunology, Autophagosomes metabolism, B-Cell CLL-Lymphoma 10 Protein chemistry, B-Cell CLL-Lymphoma 10 Protein genetics, Cell Line, Computational Biology, Computer Simulation, Mice, Models, Biological, Monte Carlo Method, Polymerization, Proteolysis, Receptors, Antigen, T-Cell metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Signal Transduction, B-Cell CLL-Lymphoma 10 Protein metabolism, Lymphocyte Activation, T-Lymphocytes immunology, T-Lymphocytes metabolism
- Abstract
The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
36. A polymer index-matched to water enables diverse applications in fluorescence microscopy.
- Author
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Han X, Su Y, White H, O'Neill KM, Morgan NY, Christensen R, Potarazu D, Vishwasrao HD, Xu S, Sun Y, Huang SY, Moyle MW, Dai Q, Pommier Y, Giniger E, Albrecht DR, Probst R, and Shroff H
- Subjects
- Animals, Microscopy, Fluorescence, Polymers, Refractometry, Caenorhabditis elegans, Water
- Abstract
We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.
- Published
- 2021
- Full Text
- View/download PDF
37. Structural and developmental principles of neuropil assembly in C. elegans.
- Author
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Moyle MW, Barnes KM, Kuchroo M, Gonopolskiy A, Duncan LH, Sengupta T, Shao L, Guo M, Santella A, Christensen R, Kumar A, Wu Y, Moon KR, Wolf G, Krishnaswamy S, Bao Z, Shroff H, Mohler WA, and Colón-Ramos DA
- Subjects
- Algorithms, Animals, Brain cytology, Brain embryology, Caenorhabditis elegans chemistry, Caenorhabditis elegans cytology, Cell Movement, Diffusion, Interneurons metabolism, Motor Neurons metabolism, Neurites metabolism, Neuropil cytology, Sensory Receptor Cells metabolism, Caenorhabditis elegans embryology, Caenorhabditis elegans metabolism, Neuropil chemistry, Neuropil metabolism
- Abstract
Neuropil is a fundamental form of tissue organization within the brain
1 , in which densely packed neurons synaptically interconnect into precise circuit architecture2,3 . However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5 . Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.- Published
- 2021
- Full Text
- View/download PDF
38. Nanoscale Pattern Extraction from Relative Positions of Sparse 3D Localizations.
- Author
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Curd AP, Leng J, Hughes RE, Cleasby AJ, Rogers B, Trinh CH, Baird MA, Takagi Y, Tiede C, Sieben C, Manley S, Schlichthaerle T, Jungmann R, Ries J, Shroff H, and Peckham M
- Subjects
- DNA, Single Molecule Imaging
- Abstract
Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.
- Published
- 2021
- Full Text
- View/download PDF
39. Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.
- Author
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Miyoshi T, Zhang Q, Miyake T, Watanabe S, Ohnishi H, Chen J, Vishwasrao HD, Chakraborty O, Belyantseva IA, Perrin BJ, Shroff H, Friedman TB, Omori K, and Watanabe N
- Subjects
- Animals, Cell Culture Techniques, Humans, Mice, Antibodies metabolism, Hybridomas metabolism, Microscopy methods, Single Molecule Imaging methods
- Abstract
Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena., Competing Interests: Declaration of interests The authors declare no conflict of interest associated with this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
40. Zebrafish Posterior Lateral Line primordium migration requires interactions between a superficial sheath of motile cells and the skin.
- Author
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Dalle Nogare DE, Natesh N, Vishwasrao HD, Shroff H, and Chitnis AB
- Subjects
- Animals, Cell Movement, Embryonic Development, Zebrafish Proteins genetics, Embryo, Nonmammalian physiology, Gene Expression Regulation, Developmental physiology, Lateral Line System embryology, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium., Competing Interests: DD, NN, HV, HS, AC No competing interests declared
- Published
- 2020
- Full Text
- View/download PDF
41. Rapid image deconvolution and multiview fusion for optical microscopy.
- Author
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Guo M, Li Y, Su Y, Lambert T, Nogare DD, Moyle MW, Duncan LH, Ikegami R, Santella A, Rey-Suarez I, Green D, Beiriger A, Chen J, Vishwasrao H, Ganesan S, Prince V, Waters JC, Annunziata CM, Hafner M, Mohler WA, Chitnis AB, Upadhyaya A, Usdin TB, Bao Z, Colón-Ramos D, La Riviere P, Liu H, Wu Y, and Shroff H
- Subjects
- Animals, Brain diagnostic imaging, Caenorhabditis elegans embryology, Cell Line, Deep Learning, Humans, Mice, Zebrafish embryology, Algorithms, Image Processing, Computer-Assisted, Microscopy
- Abstract
The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.
- Published
- 2020
- Full Text
- View/download PDF
42. Spatio-angular fluorescence microscopy III. Constrained angular diffusion, polarized excitation, and high-NA imaging.
- Author
-
Chandler T, Shroff H, Oldenbourg R, and La Rivière P
- Abstract
We investigate rotational diffusion of fluorescent molecules in angular potential wells, the excitation and subsequent emissions from these diffusing molecules, and the imaging of these emissions with high-NA aplanatic optical microscopes. Although dipole emissions only transmit six low-frequency angular components, we show that angular structured illumination can alias higher-frequency angular components into the passband of the imaging system. We show that the number of measurable angular components is limited by the relationships between three time scales: the rotational diffusion time, the fluorescence decay time, and the acquisition time. We demonstrate our model by simulating a numerical phantom in the limits of fast angular diffusion, slow angular diffusion, and weak potentials.
- Published
- 2020
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43. Sequence-Independent Self-Assembly of Germ Granule mRNAs into Homotypic Clusters.
- Author
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Trcek T, Douglas TE, Grosch M, Yin Y, Eagle WVI, Gavis ER, Shroff H, Rothenberg E, and Lehmann R
- Subjects
- Animals, Cytoplasmic Granules genetics, Drosophila Proteins genetics, Drosophila melanogaster genetics, Nuclear Proteins metabolism, Organelles physiology, RNA genetics, RNA Transport genetics, RNA, Messenger metabolism, Ribonucleoproteins genetics, Cytoplasmic Granules physiology, RNA, Messenger genetics, Ribonucleoproteins metabolism
- Abstract
mRNAs enriched in membraneless condensates provide functional compartmentalization within cells. The mechanisms that recruit transcripts to condensates are under intense study; however, how mRNAs organize once they reach a granule remains poorly understood. Here, we report on a self-sorting mechanism by which multiple mRNAs derived from the same gene assemble into discrete homotypic clusters. We demonstrate that in vivo mRNA localization to granules and self-assembly within granules are governed by different mRNA features: localization is encoded by specific RNA regions, whereas self-assembly involves the entire mRNA, does not involve sequence-specific, ordered intermolecular RNA:RNA interactions, and is thus RNA sequence independent. We propose that the ability of mRNAs to self-sort into homotypic assemblies is an inherent property of an messenger ribonucleoprotein (mRNP) that is augmented under conditions that increase RNA concentration, such as upon enrichment in RNA-protein granules, a process that appears conserved in diverse cellular contexts and organisms., Competing Interests: Declaration of Interests H.S. receives royalties, as co-inventor, from sales of iSIM by Visitech. The NIH, its officers, and staff do not recommend or endorse any company, product, or service., (Copyright © 2020 Elsevier Inc. All rights reserved.)
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- 2020
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44. WASP family proteins regulate the mobility of the B cell receptor during signaling activation.
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Rey-Suarez I, Wheatley BA, Koo P, Bhanja A, Shu Z, Mochrie S, Song W, Shroff H, and Upadhyaya A
- Subjects
- Actin-Related Protein 2-3 Complex metabolism, Actins metabolism, Animals, Antigens, CD19 metabolism, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Receptors, Antigen, B-Cell genetics, Receptors, IgG metabolism, Signal Transduction, Single Molecule Imaging, Wiskott-Aldrich Syndrome Protein Family metabolism, Wiskott-Aldrich Syndrome Protein, Neuronal genetics, B-Lymphocytes metabolism, Receptors, Antigen, B-Cell metabolism, Wiskott-Aldrich Syndrome Protein, Neuronal metabolism
- Abstract
Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of FcγRIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling.
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- 2020
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45. Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association.
- Author
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Bhagwat AR, Le Sage V, Nturibi E, Kulej K, Jones J, Guo M, Tae Kim E, Garcia BA, Weitzman MD, Shroff H, and Lakdawala SS
- Subjects
- Biological Transport, Dyneins genetics, Host-Pathogen Interactions, Humans, Influenza A virus genetics, Influenza, Human genetics, Influenza, Human virology, RNA, Viral genetics, RNA, Viral metabolism, rab GTP-Binding Proteins genetics, Dyneins metabolism, Influenza A virus physiology, Influenza, Human metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.
- Published
- 2020
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46. A 2-dimensional ratchet model describes assembly initiation of a specialized bacterial cell surface.
- Author
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Peluso EA, Updegrove TB, Chen J, Shroff H, and Ramamurthi KS
- Subjects
- Cell Membrane metabolism, Green Fluorescent Proteins, Microscopy, Fluorescence, Spores, Bacterial growth & development, Bacillus subtilis metabolism, Bacterial Proteins metabolism, Spores, Bacterial metabolism
- Abstract
Bacterial spores are dormant cells that are encased in a thick protein shell, the "coat," which participates in protecting the organism's DNA from environmental insults. The coat is composed of dozens of proteins that assemble in an orchestrated fashion during sporulation. In Bacillus subtilis , 2 proteins initiate coat assembly: SpoVM, which preferentially binds to micron-scale convex membranes and marks the surface of the developing spore as the site for coat assembly; and SpoIVA, a structural protein recruited by SpoVM that uses ATP hydrolysis to drive its irreversible polymerization around the developing spore. Here, we describe the initiation of coat assembly by SpoVM and SpoIVA. Using single-molecule fluorescence microscopy in vivo in sporulating cells and in vitro on synthetic spores, we report that SpoVM's localization is primarily driven by a lower off-rate on membranes of preferred curvature in the absence of other coat proteins. Recruitment and polymerization of SpoIVA results in the entrapment of SpoVM on the forespore surface. Using experimentally derived reaction parameters, we show that a 2-dimensional ratchet model can describe the interdependent localization dynamics of SpoVM and SpoIVA, wherein SpoVM displays a longer residence time on the forespore surface, which favors recruitment of SpoIVA to that location. Localized SpoIVA polymerization in turn prevents further sampling of other membranes by prelocalized SpoVM molecules. Our model therefore describes the dynamics of structural proteins as they localize and assemble at the correct place and time within a cell to form a supramolecular complex., Competing Interests: The authors declare no competing interest.
- Published
- 2019
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47. Spatio-angular fluorescence microscopy I. Basic theory.
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Chandler T, Shroff H, Oldenbourg R, and La Rivière P
- Abstract
We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics-a form we call the dipole spatio-angular transfer function. We discuss the implications of our analysis for all quantitative fluorescence microscopy studies and lay out a path toward a complete theory.
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- 2019
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48. Transforming the development and dissemination of cutting-edge microscopy and computation.
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Colón-Ramos DA, La Riviere P, Shroff H, and Oldenbourg R
- Subjects
- Congresses as Topic, Humans, Image Processing, Computer-Assisted methods, Microscopy methods, Microscopy standards, Molecular Imaging methods, Molecular Imaging standards
- Published
- 2019
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49. Spatio-angular fluorescence microscopy II. Paraxial 4f imaging.
- Author
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Chandler T, Shroff H, Oldenbourg R, and Rivière P
- Abstract
We investigate the properties of a single-view fluorescence microscope in a 4f geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are small when the sample contains either many randomly oriented fluorophores in each resolvable volume or unconstrained rotating fluorophores.
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- 2019
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50. Author Correction: Anchoring cortical granules in the cortex ensures trafficking to the plasma membrane for post-fertilization exocytosis.
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Vogt EJ, Tokuhiro K, Guo M, Dale R, Yang G, Shin SW, Movilla MJ, Shroff H, and Dean J
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2019
- Full Text
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