scholarly journals Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

2019 ◽  
Author(s):  
Alexander H. Clowsley ◽  
William T. Kaufhold ◽  
Tobias Lutz ◽  
Anna Meletiou ◽  
Lorenzo Di Michele ◽  
...  
Keyword(s):  
Cell Biology ◽  
Optical Resolution ◽  
Super Resolution ◽  
High Specificity ◽  
Coarse Grained ◽  
Imaging Protocol ◽  
Cellular Processes ◽  
Localization Analysis ◽  
Diffraction Imaging ◽  
Resolution Imaging

ABSTRACTInteractions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.

scholarly journals Super-Resolution Imaging With Lanthanide Luminescent Nanocrystals: Progress and Prospect

2021 ◽  
Vol 9 ◽  
Author(s):  
Hongxin Zhang ◽  
Mengyao Zhao ◽  
István M. Ábrahám ◽  
Fan Zhang
Keyword(s):  
Cell Biology ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Lanthanide Ions ◽  
Fluorescence Probes ◽  
Saturation Intensity ◽  
Resolution Imaging

Stimulated emission depletion (STED) nanoscopy has overcome a serious diffraction barrier on the optical resolution and facilitated new discoveries on detailed nanostructures in cell biology. Traditional fluorescence probes employed in the super-resolution imaging approach include organic dyes and fluorescent proteins. However, some limitations of these probes, such as photobleaching, short emission wavelengths, and high saturation intensity, still hamper the promotion of optical resolution and bio-applications. Recently, lanthanide luminescent probes with unique optical properties of non-photobleaching and sharp emissions have been applied in super-resolution imaging. In this mini-review, we will introduce several different mechanisms for lanthanide ions to achieve super-resolution imaging based on an STED-like setup. Then, several lanthanide ions used in super-resolution imaging will be described in detail and discussed. Last but not least, we will emphasize the future challenges and outlooks in hope of advancing the next-generation lanthanide fluorescent probes for super-resolution optical imaging.

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Super-Resolution Microscopy of Chromatin

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 493 ◽  
Author(s):  
Birk
Keyword(s):  
Gene Regulation ◽  
Data Analysis ◽  
Super Resolution ◽  
Fluorescence Labeling ◽  
Cellular Processes ◽  
Direct Assessment ◽  
Chromatin Interactions ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Insight Into

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals Demonstration of nanoimprinted hyperlens array for high-throughput sub-diffraction imaging

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Minseop Byun ◽  
Dasol Lee ◽  
Minkyung Kim ◽  
Yangdoo Kim ◽  
Kwan Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Medical Science ◽  
Super Resolution ◽  
Fabrication Process ◽  
Far Field ◽  
Resolution Limit ◽  
Imaging Science ◽  
Diffraction Imaging ◽  
Resolution Imaging

Abstract Overcoming the resolution limit of conventional optics is regarded as the most important issue in optical imaging science and technology. Although hyperlenses, super-resolution imaging devices based on highly anisotropic dispersion relations that allow the access of high-wavevector components, have recently achieved far-field sub-diffraction imaging in real-time, the previously demonstrated devices have suffered from the extreme difficulties of both the fabrication process and the non-artificial objects placement. This results in restrictions on the practical applications of the hyperlens devices. While implementing large-scale hyperlens arrays in conventional microscopy is desirable to solve such issues, it has not been feasible to fabricate such large-scale hyperlens array with the previously used nanofabrication methods. Here, we suggest a scalable and reliable fabrication process of a large-scale hyperlens device based on direct pattern transfer techniques. We fabricate a 5 cm × 5 cm size hyperlenses array and experimentally demonstrate that it can resolve sub-diffraction features down to 160 nm under 410 nm wavelength visible light. The array-based hyperlens device will provide a simple solution for much more practical far-field and real-time super-resolution imaging which can be widely used in optics, biology, medical science, nanotechnology and other closely related interdisciplinary fields.

Photon avalanche in lanthanide doped nanoparticles for biomedical applications: super-resolution imaging

2019 ◽  
Vol 4 (4) ◽  
pp. 881-889 ◽  
Author(s):  
Artur Bednarkiewicz ◽  
Emory M. Chan ◽  
Agata Kotulska ◽  
Lukasz Marciniak ◽  
Katarzyna Prorok
Keyword(s):  
Biomedical Applications ◽  
Super Resolution ◽  
Diffraction Imaging ◽  
Resolution Imaging

Photon avalanche in lanthanide doped nanoparticles shows exceptional properties, potentially suitable for single photoexcitation beam sub-diffraction imaging.

Visualizing membrane trafficking using total internal reflection fluorescence microscopy

2003 ◽  
Vol 31 (4) ◽  
pp. 819-823 ◽  
Author(s):  
V. Beaumont
Keyword(s):  
Fluorescence Microscopy ◽  
Membrane Trafficking ◽  
Cell Biology ◽  
Total Internal Reflection ◽  
Optical Resolution ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Live Cells ◽  
Excitable Cells ◽  
Cellular Processes

There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes, and advances in molecular biology have highlighted the ease with which proteins can now be labelled with fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy (TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane trafficking events in both non-excitable and excitable cells has been an area of notable expansion and fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the study of cellular processes at the molecular level are discussed briefly, providing promise that the future of TIR-FM in cell biology will only get brighter.

scholarly journals Translation Microscopy (TRAM) for super-resolution imaging

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhen Qiu ◽  
Rhodri S Wilson ◽  
Yuewei Liu ◽  
Alison R Dun ◽  
Rebecca S Saleeb ◽  
...  
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Ground Truth ◽  
Image Plane ◽  
Ease Of Use ◽  
Stimulated Emission ◽  
Multiple Diffraction ◽  
Microscopy Technique ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.

scholarly journals Comparing Lifeact and Phalloidin for Super-resolution Imaging of Actin in Fixed Cells

2020 ◽  
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

AbstractVisualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Label-free super-resolution imaging below 90-nm using photon-reassignment

2021 ◽  
Vol 1 ◽  
pp. 3
Author(s):  
Alberto Aguilar ◽  
Adeline Boyreau ◽  
Pierre Bon
Keyword(s):  
Biological Samples ◽  
Optical Resolution ◽  
Super Resolution ◽  
Cost Effective ◽  
Label Free ◽  
Cost Effective Approach ◽  
Imaging Mode ◽  
Resolution Imaging ◽  
Fluorescent Tags ◽  
Super Resolution Microscopy

Background: Achieving resolutions below 100 nm is key for many fields, including biology and nanomaterial characterization. Although nearfield and electron microscopy are the gold standards for studying the nanoscale, optical microscopy has seen its resolution drastically improve in the last decades. So-called super-resolution microscopy is generally based on fluorescence photophysics and requires modification of the sample at least by adding fluorescent tags, an inevitably invasive step. Therefore, it remains very challenging and rewarding to achieve optical resolutions beyond the diffraction limit in label-free samples. Methods: Here, we present a breakthrough to unlock label-free 3D super-resolution imaging of any object including living biological samples. It is based on optical photon-reassignment in confocal reflectance imaging mode. Results: We demonstrate that we surpass the resolution of all fluorescence-based confocal systems by a factor ~1.5. We have obtained images with a 3D (x,y,z) optical resolution of (86x86x248) nm3 using a visible wavelength (445 nm) and a regular microscope objective (NA=1.3). The results are presented on nanoparticles as well as on (living) biological samples. Conclusions: This cost-effective approach double the resolution of reflectance confocal microscope with minimal modifications. It is therefore compatible with any microscope and sample, works in real-time, and does not require any signal processing.

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