scholarly journals Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

2018 ◽  
Author(s):  
Guy Nir ◽  
Irene Farabella ◽  
Cynthia Pérez Estrada ◽  
Carl G. Ebeling ◽  
Brian J. Beliveau ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Imaging Data ◽  
Chromosomal Dna ◽  
Contact Maps ◽  
Integrative Modeling ◽  
Resolution Imaging ◽  
Three Dimensional Models ◽  
Single Molecule Localization Microscopy

AbstractChromosome structure is thought to be crucial for proper functioning of the nucleus. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. We begin by applying Oligopaint probes and the single-molecule localization microscopy methods of OligoSTORM and OligoDNA-PAINT to image 8 megabases of human chromosome 19, discovering that chromosomal regions contributing to compartments can form distinct structures. Intriguingly, our data also suggest that homologous maternal and paternal regions may be differentially organized. Finally, we integrate imaging data with Hi-C and restraint-based modeling using a method called integrative modeling of genomic regions (IMGR) to increase the genomic resolution of our traces to 10 kb.One Sentence SummarySuper-resolution genome tracing, contact maps, and integrative modeling enable 10 kb resolution glimpses of chromosome folding.

scholarly journals PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

scholarly journals Rapid single-wavelength lightsheet localization microscopy for clarified tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Li-An Chu ◽  
Chieh-Han Lu ◽  
Shun-Min Yang ◽  
Yen-Ting Liu ◽  
Kuan-Lin Feng ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Biological Tissues ◽  
Adult Brain ◽  
Imaging Data ◽  
Transporter Protein ◽  
Index Matching ◽  
Refractive Index Matching ◽  
Nanoscale Imaging ◽  
Resolution Imaging

Abstract Optical super-resolution microscopy allows nanoscale imaging of protein molecules in intact biological tissues. However, it is still challenging to perform large volume super-resolution imaging for entire animal organs. Here we develop a single-wavelength Bessel lightsheet method, optimized for refractive-index matching with clarified specimens to overcome the aberrations encountered in imaging thick tissues. Using spontaneous blinking fluorophores to label proteins of interest, we resolve the morphology of most, if not all, dopaminergic neurons in the whole adult brain (3.64 × 107 µm3) of Drosophila melanogaster at the nanometer scale with high imaging speed (436 µm3 per second) for localization. Quantitative single-molecule localization reveals the subcellular distribution of a monoamine transporter protein in the axons of a single, identified serotonergic Dorsal Paired Medial (DPM) neuron. Large datasets are obtained from imaging one brain per day to provide a robust statistical analysis of these imaging data.

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

scholarly journals Super resolution imaging of a distinct chromatin loop in human lymphoblastoid cells

2019 ◽  
Author(s):  
Jacqueline Jufen Zhu ◽  
Zofia Parteka ◽  
Byoungkoo Lee ◽  
Przemyslaw Szalaj ◽  
Ping Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Structure ◽  
Three Dimensional ◽  
Super Resolution ◽  
Chromatin Loop ◽  
Imaging Data ◽  
Sequencing Data ◽  
Cellular Functions ◽  
Sequencing Technologies ◽  
Chromatin Folding

AbstractThe three-dimensional genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in genomics based on sequencing technologies inferred the very basic functional chromatin folding structures of the genome known as chromatin loops, the long-range chromatin interactions that are often mediated by protein factors. To visualize the looping structure of chromatin we applied super-resolution microscopy iPALM to image a specific chromatin loop in GM12878 cells. Totally, we have generated six images of the target chromatin region at the single molecule resolution. To infer the chromatin structures from the captured images, we modeled them as looping conformations using different computational algorithms and then evaluated the models by comparing with Hi-C data to examine the concordance. The results showed a good correlation between the imaging data and sequencing data, suggesting the visualization of higher-order chromatin structures for the very short genomic segments can be realized by microscopic imaging.

scholarly journals Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling

PLoS Genetics ◽  
2018 ◽  
Vol 14 (12) ◽  
pp. e1007872 ◽  
Author(s):  
Guy Nir ◽  
Irene Farabella ◽  
Cynthia Pérez Estrada ◽  
Carl G. Ebeling ◽  
Brian J. Beliveau ◽  
...  
Keyword(s):  
Super Resolution ◽  
Contact Maps ◽  
Integrative Modeling ◽  
Resolution Imaging

scholarly journals Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Adrien C. Descloux ◽  
Kristin S. Grußmayer ◽  
Aleksandra Radenovic
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Free Resolution ◽  
Temporal Separation ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Localization Precision ◽  
Microscopy Data ◽  
Resolution Estimation ◽  
Single Molecule Localization Microscopy

AbstractLocalization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

scholarly journals Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

2021 ◽  
Vol 13 ◽  
Author(s):  
Gabriella Gagliano ◽  
Tyler Nelson ◽  
Nahima Saliba ◽  
Sofía Vargas-Hernández ◽  
Anna-Karin Gustavsson
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

scholarly journals Volumetric super-resolution imaging by serial ultrasectioning and STochastic Optical Reconstruction Microscopy (STORM) in neural tissue

2021 ◽  
Author(s):  
Tarlan Vatan ◽  
Jacqueline A. Minehart ◽  
Chenghang Zhang ◽  
Vatsal Agarwal ◽  
Jerry Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Neural Tissue ◽  
Imaging Data ◽  
Whole Cells ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Tissue Scattering ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Chemical Synapses

Abstract Here we present a protocol for collecting large-volume, four-color, single-molecule localization imaging data from neural tissue. We have applied this technique to map the location and identities of chemical synapses across whole cells in mouse retinae. Our sample preparation approach improves 3D STORM image quality by reducing tissue scattering, photobleaching, and optical distortions associated with deep imaging. This approach can be extended for use on other tissue types enabling life scientists to perform volumetric super-resolution imaging in diverse biological models. For a detailed application of this protocol, please refer to Sigal et al., 2015.

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