A System-level Approach to Single-Molecule Live-Cell Fluorescence Microscopy

Harriman, Oliver; Leake, Mark C.

doi:10.22443/rms.inf.1.93

Cited by 2 publications

(2 citation statements)

References 9 publications

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“…a few tenths of a mm). Advanced light microscopy systems are now being designed using systems engineering principles to relate directly to the specifics of different biological processes [462,463].…”

Section: Probing Single Molecules In Populations Of Cells and Tissuesmentioning

confidence: 99%

Single-molecule techniques in biophysics: a review of the progress in methods and applications

et al. 2017

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Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k T, where k is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

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Section: Probing Single Molecules In Populations Of Cells and Tissuesmentioning

confidence: 99%

Single-molecule techniques in biophysics: a review of the progress in methods and applications

et al. 2017

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“…Here we report recent advances of single-molecule optical proteomics in unicellular organisms, enabling insight at 'bottom-up' molecular scales, and associated developments required for the new biophysical technology which, in itself, can be designed using systems engineering principles informed by underlying biological processes (21).…”

Section: Introductionmentioning

confidence: 99%

Systems biophysics: Single-molecule optical proteomics in single living cells

Shashkova

Leake

2018

Current Opinion in Systems Biology

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How does the interplay between biomolecules result in the emergence of cellular complexity at higher length scales? This interplay in even simple biological processes is often too challenging to probe using traditional experimental tools of ensemble averaging across several thousands of molecules.Instead, insight can be gained using single-molecule techniques which can unpick the heterogeneity in physical/chemical properties of biomolecules and their cellular interactions. Significant understanding of many biological systems can be gained using techniques which apply advanced fluorescence microscopy to determine the cellular localization, dynamics and interaction kinetics of single functional proteins, whilst retaining the native context of live cells. Here, we report recent advances applied to cell motility, DNA replication and gene regulation in model unicellular organisms. Highlights Single-molecule optical proteomics can probe whole biological systems  Several processes (motility, DNA replication, gene regulation) have been probed  Real-time readout of molecular systems can be probed in model unicellular organisms

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A System-level Approach to Single-Molecule Live-Cell Fluorescence Microscopy

Cited by 2 publications

References 9 publications

Single-molecule techniques in biophysics: a review of the progress in methods and applications

Single-molecule techniques in biophysics: a review of the progress in methods and applications

Systems biophysics: Single-molecule optical proteomics in single living cells

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