Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging

Yang, Zhongtian; Li, Liuju; Ling, Jing; Liu, Tianyan; Huang, Xiaoshuai; Ying, Yuqing; Zhao, Yun; Zhao, Yan; Lei, Kai Y.; Chen, Liangyi; Chen, Zhuo

doi:10.1039/d0sc02837a

Cited by 66 publications

(66 citation statements)

References 58 publications

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“…32 As well as adding triplet state quenchers (TSQ) and oxygen-savaging systems into the solution, TSQs including cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) have been covalently conjugated to the fluorophore. 32,33 This is a solution to fast quenching rate and especially suitable for long observation time.…”

Section: Experimental Designmentioning

confidence: 99%

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Yang

Wang

et al. 2021

Appl Spectrosc

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Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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Section: Experimental Designmentioning

confidence: 99%

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Yang

Wang

et al. 2021

Appl Spectrosc

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“…Some components of the medium such as phenol red, fetal bovine serum, riboflavins and vitamins can produce substantial fluorescence background signal, limiting the ability to detect the signal of interest and impacting the accuracy and precision of quantitative measurements. Additionally, the concentration of a fluorescent dye and the solvent used or transfection reagents and expression of fluorescent protein fusions 37 in a live sample may affect cell function [40][41][42] , induce synergistic effects with the conditions used (for example, drug treatments), and therefore impact reproducibility.…”

Section: Guidelines On Reporting Sample Preparationmentioning

confidence: 99%

Best practices and tools for reporting reproducible fluorescence microscopy methods

et al. 2021

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Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.

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“…Among these techniques, SIM excels in extending the spatial resolution with a relatively small increase in emission photons [7]. Thus, SIM is suitable for live-cell SR imaging, such as identification of vesicle fusion intermediates including enlarged fusion pores by 300 Hz SR imaging, 1 h time-lapse SR imaging of actin filaments labelled by lifeact-EGFP with minimal photobleaching [8], and 2000-frame continuous mitochondrial SR imaging with small enlargements of cristae diameters [9]. Despite these advantages, SIM is prone to reconstruction artefacts [10].…”

Section: Introductionmentioning

confidence: 99%

Structured illumination microscopy artefacts caused by illumination scattering

Fang

Fan

et al. 2021

Phil. Trans. R. Soc. A.

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Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artefacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artefacts caused by scattering in thick samples. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.

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Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging

Abstract: Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies...

Cited by 66 publications

References 58 publications

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Best practices and tools for reporting reproducible fluorescence microscopy methods

Structured illumination microscopy artefacts caused by illumination scattering

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