Extracting quantitative information from single-molecule super-resolution imaging data with LAMA – LocAlization Microscopy Analyzer

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.

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Rapid single-wavelength lightsheet localization microscopy for clarified tissue

Nature Communications ◽

10.1038/s41467-019-12715-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 3

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.

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

10.1101/374058 ◽

2018 ◽

Cited By ~ 2

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.

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Imaging unlabeled proteins on DNA with super-resolution

Nucleic Acids Research ◽

10.1093/nar/gkaa061 ◽

2020 ◽

Vol 48 (6) ◽

pp. e34-e34 ◽

Cited By ~ 1

Author(s):

Anna E C Meijering ◽

Andreas S Biebricher ◽

Gerrit Sitters ◽

Ineke Brouwer ◽

Erwin J G Peterman ◽

...

Keyword(s):

Detection Limit ◽

Single Molecule ◽

Super Resolution ◽

Diffraction Limit ◽

Current Image ◽

Localization Microscopy ◽

Resolution Imaging ◽

Biomolecular Analysis ◽

Current Detection ◽

Inverse Imaging

Abstract Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or ‘iBALM’ can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

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

Communications Biology ◽

10.1038/s42003-021-02086-1 ◽

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.

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

10.21203/rs.3.pex-1587/v1 ◽

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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Quantitative Analysis of Nuclear Lamins Imaged by Super-Resolution Light Microscopy

Cells ◽

10.3390/cells8040361 ◽

2019 ◽

Vol 8 (4) ◽

pp. 361 ◽

Cited By ~ 5

Author(s):

Mark Kittisopikul ◽

Laura Virtanen ◽

Pekka Taimen ◽

Robert D. Goldman

Keyword(s):

Light Microscopy ◽

Single Molecule ◽

Electron Tomography ◽

Super Resolution ◽

Nuclear Lamina ◽

Quantitative Information ◽

Structured Illumination ◽

Imaging Data ◽

Structured Illumination Microscopy ◽

Nuclear Lamins

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

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A H-bond strategy to develop acid-resistant photoswitchable rhodamine spirolactams for super-resolution single-molecule localization microscopy

Chemical Science ◽

10.1039/c9sc01284b ◽

2019 ◽

Vol 10 (18) ◽

pp. 4914-4922 ◽

Cited By ~ 26

Author(s):

Qingkai Qi ◽

Weijie Chi ◽

Yuanyuan Li ◽

Qinglong Qiao ◽

Jie Chen ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Amino Groups ◽

Localization Microscopy ◽

Resolution Imaging ◽

Resolution Single ◽

Single Molecule Localization Microscopy

Rhodamine spirolactams with adjacent amino groups work as acid-resistant and photoswitchable fluorophores in single-molecule localization super-resolution imaging.

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Visualization and intracellular dynamic tracking of exosomes and exosomal miRNAs using single molecule localization microscopy

Nanoscale ◽

10.1039/c7nr08800k ◽

2018 ◽

Vol 10 (11) ◽

pp. 5154-5162 ◽

Cited By ~ 16

Author(s):

Chen Chen ◽

Shenfei Zong ◽

Zhuyuan Wang ◽

Ju Lu ◽

Dan Zhu ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Localization Microscopy ◽

Dynamic Tracking ◽

Exosomal Mirnas ◽

Resolution Imaging ◽

Intracellular Dynamic ◽

Single Molecule Localization Microscopy

Super-resolution imaging and dynamic tracking of cancer-derived exosomes and exosomal miRNAs were realized using single molecule localization microscopy.

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