Super-resolution fluorescent materials: an insight into design and bioimaging applications

2016 ◽  
Vol 45 (17) ◽  
pp. 4651-4667 ◽  
Author(s):  
Zhigang Yang ◽  
Amit Sharma ◽  
Jing Qi ◽  
Xiao Peng ◽  
Dong Yeop Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Organic Fluorophores ◽  
Biological Targets ◽  
Fluorescent Materials ◽  
Microscopy Techniques ◽  
Insight Into

With the emerging of super-resolution fluorescent imaging microscopy techniques, biological targets below 200 nm in size are successful to be localized clearly and precisely with unprecedented details. In this tutorial review, the fluorescent materials, including organic fluorophores and nanomaterials, utilized in STED, single molecule localized microscopy (PALM/STORM) and SOFI microscopies, together with their working principles are mainly discussed.

scholarly journals Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

2021 ◽  
Author(s):  
Matthew D. Lycas ◽  
Aske L. Ejdrup ◽  
Andreas T. Sørensen ◽  
Nicolai O. Haahr ◽  
Søren H. Jørgensen ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Striatal Slices ◽  
D2 Autoreceptor ◽  
Molecular Components ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Excitatory Drive

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

Solid-State Nanopores: From Fabrication to Application

2012 ◽  
Vol 20 (5) ◽  
pp. 24-29 ◽  
Author(s):  
Adam R. Hall
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
High Speed ◽  
Super Resolution ◽  
Magnetic Tweezers ◽  
Fluorescent Imaging ◽  
Force Microscopy ◽  
Atomic Force ◽  
Labeling Requirements ◽  
Molecular Manipulation

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

scholarly journals Precision of fiducial marker alignment for correlative super‐resolution fluorescence and transmission electron microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Adeeba Fathima ◽  
César Augusto Quintana-Cataño ◽  
Christoph Heintze ◽  
Michael Schlierf
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Specific Protein ◽  
Fiducial Markers ◽  
Transmission Electron ◽  
Microscopy Techniques

AbstractRecent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

scholarly journals On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

2019 ◽  
Vol 21 (7) ◽  
pp. 3721-3733 ◽  
Author(s):  
Jochem H. Smit ◽  
Jasper H. M. van der Velde ◽  
Jingyi Huang ◽  
Vanessa Trauschke ◽  
Sarah S. Henrikus ◽  
...  
Keyword(s):  
Triplet State ◽  
Single Molecule ◽  
Photophysical Properties ◽  
Super Resolution ◽  
Organic Fluorophores ◽  
Resolution Imaging ◽  
The Impact ◽  
Kinetics Of

How photostabilizer molecules influence the photophysical properties of various organic fluorophores used for single-molecule and super-resolution imaging.

Fluorescence Spectroscopy and Microscopy Techniques

2018 ◽  
pp. 27-66
Author(s):  
David Bensimon ◽  
Vincent Croquette ◽  
Jean-François Allemand ◽  
Xavier Michalet ◽  
Terence Strick
Keyword(s):  
Single Molecule ◽  
Energy Levels ◽  
Diffusion Constant ◽  
Super Resolution ◽  
Noise Signal ◽  
Single Molecule Fluorescence ◽  
Fluorescence Detector ◽  
Polarization Spectroscopy ◽  
Mechanical Explanation ◽  
Microscopy Techniques

This chapter reviews the use of fluorescent methods in the study of single molecules, how the foundations of fluorescence are rooted in Einstein’s description of absorption and emission (spontaneous and stimulated), and their quantum-mechanical explanation in terms of transitions between quantized energy levels, as represented in Jablonski diagrams. It describes the non-radiative channels which compete with fluorescent emission, decrease its efficiency, and ultimately destroy the fluorescent molecule. Fluorescence Polarization Spectroscopy, FRET, and FCS are briefly presented. Without reviewing the various available fluorophores, it describes the various illumination methods used to study them, sketching super-resolution methods (STED, STORM, PALM) that have recently allowed fluorophores to be resolved to a few tens of nanometres. Finally, it describes the considerations (bandwidth, signal to noise, signal to background) used in choosing a single-molecule fluorescence detector, and the extraction of the diffusion constant of a fluorophore from the finite time, noisy traces of its positions.

scholarly journals Beginner’s guide to producing super-resolved images on a widefield fluorescence microscope

2020 ◽  
Vol 42 (4) ◽  
pp. 52-56
Author(s):  
Ilijana Vojnovic ◽  
Ulrike Endesfelder
Keyword(s):  
Fluorescence Microscopy ◽  
Sample Preparation ◽  
Single Molecule ◽  
Life Sciences ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Specialized Hardware ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

The development of super-resolution microscopy techniques, which are able to achieve resolutions in the nanometre range and as such allow the visualization of subcellular structures and dynamics, has considerably expanded the possibilities of fluorescence microscopy in the life sciences. While a majority of these techniques require highly specialized hardware, single-molecule localization microscopy (SMLM) can be implemented on conventional widefield fluorescence microscopes. Here, we describe what technical upgrades are necessary and discuss some of the difficulties that can be encountered during sample preparation and imaging.

scholarly journals Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

2020 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Joshua C. Price ◽  
Raquel Mesquita-Riberio ◽  
Melissa L. Mather ◽  
Izzy Jayasinghe
Keyword(s):  
Single Molecule ◽  
Light Exposure ◽  
Super Resolution ◽  
Observation Time ◽  
Nitrogen Vacancy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Nitrogen Vacancy Centers ◽  
Optical Reconstruction ◽  
Living Neurons ◽  
Microscopy Techniques

AbstractStochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the ‘off’-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at ∼ 3-times the speed and ∼ 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

scholarly journals Self-healing dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Jasper H. M. van der Velde ◽  
Jochem Smit ◽  
Michiel Punter ◽  
Thorben Cordes
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Superior Performance ◽  
Self Healing ◽  
Sted Microscopy ◽  
Performance Results ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Oligonucleotide Conjugates ◽  
Kinetics Of

AbstractIn recent years optical microscopy techniques have emerged that allow optical imaging at unprecented resolution beyond the diffraction limit. Up to date, photostabilizing buffers are the method of choice to realize either photoswitching and/or to enhance the signal brightness and stability of the employed fluorescent probes. This strategy has, however, restricted applicability and is not suitable for live cell imaging. In this paper, we tested the performance of self-healing organic fluorophores with intramolecular photostabilization in super-resolution microscopy with targeted (STED) and stochastic readout (STORM). The overall goal of the study was to improve the spatial and temporal resolution of both techniques without the need for mixtures of photostabilizing agents in the imaging buffer. Due to its past superior performance we identified ATTO647N-photostabilizer conjugates as suitable candidates for STED microscopy. We characterize the photostability and resulting performance of NPA-ATTO647N oligonucleotide conjugates in STED microscopy. We find that the superior photophysical performance results in optimal STED imaging and demonstrate the possibility to obtain single-molecule fluorescent transients of individual fluorophores while illuminating with both the excitation- and STED-laser. Secondly, we show an analysis of photoswitching kinetics of self-healing Cy5 dyes (comprising TX, COT and NPA stabilizers) in the presence of TCEP- and cysteamine, which are typically used in STORM microscopy. In line with previous work, we find that intramolecular photostabilization strongly influences photoswitching kinetics and requires careful attention when designing STORM-experiments. In summary, this contribution explores the possibilities and limitations of self-healing dyes in super-resolution microscopy of differing modalities.

scholarly journals Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

2014 ◽  
Vol 25 (1) ◽  
pp. 15 ◽  
Author(s):  
Isuru D. Jayasinghe ◽  
Alexander H. Clowsley ◽  
Michelle Munro ◽  
Yufeng Hou ◽  
David J. Crossman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Single Molecule ◽  
Striated Muscle ◽  
Calcium Signalling ◽  
Super Resolution ◽  
Muscle Cells ◽  
Muscle Fibres ◽  
T Tubules ◽  
Microscopy Techniques

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

ChemInform Abstract: Super-Resolution Fluorescent Materials: An Insight into Design and Bioimaging Applications

ChemInform ◽  
2016 ◽  
Vol 47 (42) ◽  
Author(s):  
Zhigang Yang ◽  
Amit Sharma ◽  
Jing Qi ◽  
Xiao Peng ◽  
Dong Yeop Lee ◽  
...  
Keyword(s):  
Super Resolution ◽  
Fluorescent Materials ◽  
Insight Into
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