scholarly journals RhoBAST - a rhodamine-binding aptamer for super-resolution RNA imaging

2020 ◽  
Author(s):  
Murat Sunbul ◽  
Jens Lackner ◽  
Annabell Martin ◽  
Daniel Englert ◽  
Benjamin Hacene ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Image Resolution ◽  
Rna Aptamer ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Fluorescence Light ◽  
Rhodamine Dye ◽  
Single Molecule Localization Microscopy

AbstractRhoBAST is a novel fluorescence light-up RNA aptamer (FLAP) that transiently binds a fluorogenic rhodamine dye. Fast dye association and dissociation result in intermittent fluorescence emission, facilitating single-molecule localization microscopy (SMLM) with an image resolution not limited by photobleaching. We demonstrate RhoBAST's excellent properties as a RNA marker by resolving subcellular and subnuclear structures of RNA in live and fixed cells by SMLM and structured illumination microscopy (SIM).

scholarly journals Resolving the internal morphology of core–shell microgels with super-resolution fluorescence microscopy

2020 ◽  
Vol 2 (1) ◽  
pp. 323-331 ◽  
Author(s):  
Pia Otto ◽  
Stephan Bergmann ◽  
Alice Sandmeyer ◽  
Maxim Dirksen ◽  
Oliver Wrede ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Internal Structure ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Core Shell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Internal Morphology

We investigate the internal structure of smart core–shell microgels by super-resolution fluorescence microscopy by combining of 3D single molecule localization and structured illumination microscopy using freely diffusing fluorescent dyes.

scholarly journals Drosophila germ granules are structured and contain homotypic mRNA clusters

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Tatjana Trcek ◽  
Markus Grosch ◽  
Andrew York ◽  
Hari Shroff ◽  
Timothée Lionnet ◽  
...  
Keyword(s):  
Single Molecule ◽  
Germ Plasm ◽  
Cell Formation ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Germ Granules ◽  
Spatially Distributed ◽  
Multiple Copies ◽  
And Migration

Abstract Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters’ that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules.

scholarly journals Laser-free super-resolution microscopy

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Meiotic Chromosomes ◽  
Set Up ◽  
Super Resolution Microscopy

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as ‘blinking’), detected and localized. The use of a short burst of deep blue excitation (350–380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

scholarly journals Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope

2021 ◽  
Vol 379 (2199) ◽  
Author(s):  
Christian Pilger ◽  
Jakub Pospíšil ◽  
Marcel Müller ◽  
Martin Ruoff ◽  
Martin Schütte ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Super Resolution ◽  
Image Resolution ◽  
Laser Scanning Microscopy ◽  
Tissue Imaging ◽  
Source Image ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Fluorescence Excitation ◽  
Two Photon

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

scholarly journals Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy

2018 ◽  
Author(s):  
Hesam Mazidi ◽  
Jin Lu ◽  
Arye Nehorai ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Estimation Algorithm ◽  
Image Resolution ◽  
Spread Function ◽  
Localization Errors ◽  
Imaging Artifacts ◽  
Detection And Localization ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

ABSTRACTSingle-molecule localization microscopy (SMLM) depends on sequential detection and localization of individual molecular blinking events. Due to the stochasticity of single-molecule blinking and the desire to improve SMLM’s temporal resolution, algorithms capable of analyzing frames with a high density (HD) of active molecules, or molecules whose images overlap, are a prerequisite for accurate location measurements. Thus far, HD algorithms are evaluated using scalar metrics, such as root-mean-square error, that fail to quantify the structure of errors caused by the structure of the sample. Here, we show that the spatial distribution of localization errors within super-resolved images of biological structures are vectorial in nature, leading to systematic structural biases that severely degrade image resolution. We further demonstrate that the shape of the microscope’s point-spread function (PSF) fundamentally affects the characteristics of imaging artifacts. We built a Robust Statistical Estimation algorithm (RoSE) to minimize these biases for arbitrary structures and PSFs. RoSE accomplishes this minimization by estimating the likelihood of blinking events to localize molecules more accurately and eliminate false localizations. Using RoSE, we measure the distance between crossing microtubules, quantify the morphology of and separation between vesicles, and obtain robust recovery using diverse 3D PSFs with unmatched accuracy compared to state-of-the-art algorithms.

scholarly journals Super-Resolution Imaging of Tight and Adherens Junctions: Challenges and Open Questions

2020 ◽  
Vol 21 (3) ◽  
pp. 744 ◽  
Author(s):  
Hannes Gonschior ◽  
Volker Haucke ◽  
Martin Lehmann
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Rapid Development ◽  
Ion Homeostasis ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Molecular Architecture ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Resolution Imaging

The tight junction (TJ) and the adherens junction (AJ) bridge the paracellular cleft of epithelial and endothelial cells. In addition to their role as protective barriers against bacteria and their toxins they maintain ion homeostasis, cell polarity, and mechano-sensing. Their functional loss leads to pathological changes such as tissue inflammation, ion imbalance, and cancer. To better understand the consequences of such malfunctions, the junctional nanoarchitecture is of great importance since it remains so far largely unresolved, mainly because of major difficulties in dynamically imaging these structures at sufficient resolution and with molecular precision. The rapid development of super-resolution imaging techniques ranging from structured illumination microscopy (SIM), stimulated emission depletion (STED) microscopy, and single molecule localization microscopy (SMLM) has now enabled molecular imaging of biological specimens from cells to tissues with nanometer resolution. Here we summarize these techniques and their application to the dissection of the nanoscale molecular architecture of TJs and AJs. We propose that super-resolution imaging together with advances in genome engineering and functional analyses approaches will create a leap in our understanding of the composition, assembly, and function of TJs and AJs at the nanoscale and, thereby, enable a mechanistic understanding of their dysfunction in disease.

scholarly journals Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

2020 ◽  
Author(s):  
Lucia C. S. Wunderlich ◽  
Florian Ströhl ◽  
Stefan Ströhl ◽  
Oliver Vanderpoorten ◽  
Luca Mascheroni ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Single Molecule ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Glomerular Filtration Barrier ◽  
Maximum Resolution ◽  
Filtration Barrier ◽  
Super Resolution Microscopy

AbstractImmunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.

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

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 361 ◽  
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.

scholarly journals Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy

2019 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Marie-Christin Spindler ◽  
Sebastian Reinhard ◽  
Teresa Klein ◽  
Andreas Kurz ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Single Molecule ◽  
Super Resolution ◽  
Structural Data ◽  
Molecular Organization ◽  
Molecular Architecture ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Automatic Image Processing ◽  
Ultrastructure Preservation

AbstractThe synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different ExM protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased expansion factor determination. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20-30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

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