Quantitative microscopy based on single-molecule fluorescence

Jung, Seung‐Ryoung; Fujimoto, Bryant S.; Chiu, Daniel T.

doi:10.1016/j.cbpa.2017.06.004

Cited by 16 publications

(22 citation statements)

References 48 publications

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“…Super-resolution imaging of RNaseE and ribosomal proteins ( Fig 5D) suggest that this energetic barrier exists for BR-bodies, as well (18). (133,134). A uniform or random spatial distribution of components within a compartment can also provide a benchmark for LLPS (1).…”

Section: Super-resolution Microscopy As a Tool To Assess Llps In Bactmentioning

confidence: 98%

The emergence of phase separation as an organizing principle in bacteria

Azaldegui

Vecchiarelli

Biteen

2020

Preprint

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Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria.Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.

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Section: Super-resolution Microscopy As a Tool To Assess Llps In Bactmentioning

confidence: 98%

The emergence of phase separation as an organizing principle in bacteria

Azaldegui

Vecchiarelli

Biteen

2020

Preprint

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“…A major advantage of the approach is its potential for use in living cells, especially for targets which are difficult-to-reach by extrinsic fluorescent markers or have precluded SMLM read-out channels due to low signal-to-noise/high background, e.g., arising from intracellular autofluorescence. Like all (sequential) SMLM techniques, our approach is limited by the temporal resolution needed for structural studies of abundant proteins but offers the read-out of two different proteins by intrinsic FP labels with excellent SMLM properties to measure their molecule dynamics within the same compartment or cell [ 25 ].…”

Section: Resultsmentioning

confidence: 99%

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Virant

Turkowyd

Balinovic

et al. 2017

IJMS

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Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes.

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“…To convert fluorescence-based estimates into protein numbers not only autofluorescence and unspecific labeling must be considered by additional controls and calibration experiments but also labeling efficiency and stoichiometry as well as potential interference with sample structures. Among the most prominent SMFS counting methods [112][113][114][115][116] are fluorescence intensity quantification [8], photobleaching step counting [11,117,118], stochastic single-molecule based super-resolution microscopy (SMLM [10,[119][120][121], bSOFI [122] & qPAINT [123]), and quantification based on photon antibunching [124,125] (CoPS).…”

Section: Sample Preparationmentioning

confidence: 99%

Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting

Gruβmayer

Yserentant²,

Herten³

2019

Methods Appl. Fluoresc.

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The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in superresolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.

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Quantitative microscopy based on single-molecule fluorescence

Cited by 16 publications

References 48 publications

The emergence of phase separation as an organizing principle in bacteria

The emergence of phase separation as an organizing principle in bacteria

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting

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