scholarly journals Extending the spatiotemporal resolution of super-resolution microscopies using photomodulatable fluorescent proteins

2016 ◽  
Vol 09 (03) ◽  
pp. 1630009 ◽  
Author(s):  
Mingshu Zhang ◽  
Zhifei Fu ◽  
Pingyong Xu
Keyword(s):  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Fluorescence Emission ◽  
Optical Resolution ◽  
Super Resolution ◽  
Time Lapse ◽  
Diffraction Limit ◽  
Spatiotemporal Resolution ◽  
The Past ◽  
Microscopy Techniques

In the past two decades, various super-resolution (SR) microscopy techniques have been developed to break the diffraction limit using subdiffraction excitation to spatially modulate the fluorescence emission. Photomodulatable fluorescent proteins (FPs) can be activated by light of specific wavelengths to produce either stochastic or patterned subdiffraction excitation, resulting in improved optical resolution. In this review, we focus on the recently developed photomodulatable FPs or commonly used SR microscopies and discuss the concepts and strategies for optimizing and selecting the biochemical and photophysical properties of PMFPs to improve the spatiotemporal resolution of SR techniques, especially time-lapse live-cell SR techniques.

scholarly journals Fluorescent Probes for STED Optical Nanoscopy

Nanomaterials ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 21
Author(s):  
Sejoo Jeong ◽  
Jerker Widengren ◽  
Jong-Chan Lee
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Optical Resolution ◽  
Super Resolution ◽  
Fluorescent Nanoparticles ◽  
Resolution Limit ◽  
Photochemical Properties ◽  
Super Resolution Microscopy

Progress in developing fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, is inseparable from the advancement in optical fluorescence microscopy. Super-resolution microscopy, or optical nanoscopy, overcame the far-field optical resolution limit, known as Abbe’s diffraction limit, by taking advantage of the photophysical properties of fluorescent probes. Therefore, fluorescent probes for super-resolution microscopy should meet the new requirements in the probes’ photophysical and photochemical properties. STED optical nanoscopy achieves super-resolution by depleting excited fluorophores at the periphery of an excitation laser beam using a depletion beam with a hollow core. An ideal fluorescent probe for STED nanoscopy must meet specific photophysical and photochemical properties, including high photostability, depletability at the depletion wavelength, low adverse excitability, and biocompatibility. This review introduces the requirements of fluorescent probes for STED nanoscopy and discusses the recent progress in the development of fluorescent probes, such as fluorescent proteins, organic dyes, and fluorescent nanoparticles, for the STED nanoscopy. The strengths and the limitations of the fluorescent probes are analyzed in detail.

scholarly journals Super-resolution microscopy: successful applications in centrosome study and beyond

2019 ◽  
Vol 5 (5-6) ◽  
pp. 235-243 ◽  
Author(s):  
Jingyan Fu ◽  
Chuanmao Zhang
Keyword(s):  
Molecular Basis ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
The Past ◽  
Eukaryotic Species ◽  
Organizing Center ◽  
Cilia And Flagella ◽  
Super Resolution Microscopy

AbstractCentrosome is the main microtubule-organizing center in most animal cells. Its core structure, centriole, also assembles cilia and flagella that have important sensing and motility functions. Centrosome has long been recognized as a highly conserved organelle in eukaryotic species. Through electron microscopy, its ultrastructure was revealed to contain a beautiful nine-symmetrical core 60 years ago, yet its molecular basis has only been unraveled in the past two decades. The emergence of super-resolution microscopy allows us to explore the insides of a centrosome, which is smaller than the diffraction limit of light. Super-resolution microscopy also enables the compartmentation of centrosome proteins into different zones and the identification of their molecular interactions and functions. This paper compiles the centrosome architecture knowledge that has been revealed in recent years and highlights the power of several super-resolution techniques.

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 Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2019 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

scholarly journals Super-Resolution Imaging with Graphene

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 307
Author(s):  
Xiaoxiao Jiang ◽  
Lu Kong ◽  
Yu Ying ◽  
Qiongchan Gu ◽  
Jiangtao Lv ◽  
...  
Keyword(s):  
Optical Imaging ◽  
Near Field ◽  
Super Resolution ◽  
High Resolution Imaging ◽  
The Past ◽  
Working Principle ◽  
Resolution Imaging ◽  
Conventional Microscopy ◽  
Microscopy Techniques ◽  
Research Domain

Super-resolution optical imaging is a consistent research hotspot for promoting studies in nanotechnology and biotechnology due to its capability of overcoming the diffraction limit, which is an intrinsic obstacle in pursuing higher resolution for conventional microscopy techniques. In the past few decades, a great number of techniques in this research domain have been theoretically proposed and experimentally demonstrated. Graphene, a special two-dimensional material, has become the most meritorious candidate and attracted incredible attention in high-resolution imaging domain due to its distinctive properties. In this article, the working principle of graphene-assisted imaging devices is summarized, and recent advances of super-resolution optical imaging based on graphene are reviewed for both near-field and far-field applications.

scholarly journals Embracing the uncertainty: the evolution of SOFI into a diverse family of fluctuation-based super-resolution microscopy methods

2021 ◽  
Author(s):  
Monika Pawlowska ◽  
Ron Tenne ◽  
Bohnishikha Ghosh ◽  
Adrian Makowski ◽  
Radek Lapkiewicz
Keyword(s):  
3D Imaging ◽  
Deep Neural Networks ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Significant Progress ◽  
Spatial Correlations ◽  
Order Of Magnitude ◽  
Temporal And Spatial ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy techniques have pushed the limits of resolution in optical imaging by more than an order of magnitude. However, these methods often require long acquisition times as well as complex setups and sample preparation protocols. Super-resolution Optical Fluctuation Imaging (SOFI) emerged over ten years ago as an approach that exploits temporal and spatial correlations within the acquired images to obtain increased resolution with less strict requirements. This review follows the progress of SOFI from its first demonstration to the development of a branch of methods that treat fluctuations as a source of contrast, rather than noise. Among others, we highlight the implementation of SOFI with standard fluorescent proteins as well as the microscope modification that facilitate 3D imaging and the application of modern cameras. Going beyond the classical framework of SOFI, we explore different innovative concepts from deep neural networks all the way to a quantum analogue of SOFI, antibunching microscopy. While SOFI has not reached the same level of ubiquity as other super-resolution methods, our overview finds significant progress and substantial potential for the concept of leveraging fluorescence fluctuations to obtain super-resolved images.

scholarly journals A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Microscopy ◽  
2021 ◽  
Author(s):  
Tetsuichi Wazawa ◽  
Ryohei Noma ◽  
Shusaku Uto ◽  
Kazunori Sugiura ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Dependence ◽  
Super Resolution ◽  
Time Lapse ◽  
Light Irradiation ◽  
Acquisition Time ◽  
Polarization Angle ◽  
Cellular Dynamics

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

scholarly journals Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Yingying Jing ◽  
Chenshuang Zhang ◽  
Bin Yu ◽  
Danying Lin ◽  
Junle Qu
Keyword(s):  
Biological Activities ◽  
Super Resolution ◽  
Spatiotemporal Resolution ◽  
The Past ◽  
Relevant Field ◽  
Living Species ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Conventional Light Microscopy

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.

scholarly journals SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

2019 ◽  
Author(s):  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
Peter Dedecker
Keyword(s):  
Fluorescence Imaging ◽  
Computational Analysis ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Imaging Data ◽  
Sample Structure ◽  
Imaging Conditions

AbstractSuper-resolution fluorescence imaging techniques allow optical imaging of specimens beyond the diffraction limit of light. Super-resolution optical fluctuation imaging (SOFI) relies on computational analysis of stochastic blinking events to obtain a super-resolved image. As with some other super-resolution methods, this strong dependency on computational analysis can make it difficult to gauge how well the resulting images reflect the underlying sample structure. We herein report SOFIevaluator, an unbiased and parameter-free algorithm for calculating a set of metrics that describes the quality of super-resolution fluorescence imaging data for SOFI. We additionally demonstrate how SOFIevaluator can be used to identify fluorescent proteins that perform well for SOFI imaging under different imaging conditions.

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.

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