Using bespoke fluorescence microscopy to study the soft condensed matter of living cells at the single molecule level

Xue, Qing; Harriman, Oliver; Leake, Mark C.

doi:10.1088/1742-6596/286/1/012001

Cited by 3 publications

(3 citation statements)

References 11 publications

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“…The methods outlined here illustrate not only how light microscopy has evolved from a qualitative observational tool into a highly quantitative instrument, 23 but also how bespoke tools from physics can be developed to characterize properties of the living component of soft-matter at the single-molecule length scale 42 not just at an controlled, reductionist in vitro level 43 but also to gain molecular-level insight into the physiologically relevant context of single functioning, living cells. [44][45][46][47][48][49] Combining this automated CoPro method with multicolour single-molecule real-time fluorescence imaging 50,51 may also enable quantitative estimation of dynamic protein concentration changes of multiple interacting proteins in live cells, which is an appealing route towards investigating native biochemistry, one molecule at a time.…”

Section: Discussionmentioning

confidence: 99%

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

2015

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We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of protein concentrations in single functional cells, and enables the distributions of concentrations across a cell population to be measured. This could be useful in investigating several cellular processes that are mediated by proteins, especially where changes in protein concentration in a single cell in response to changes in the extracellular chemical environment are subtle and rapid and may be smaller than the variability across a cell population.

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Section: Discussionmentioning

confidence: 99%

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

2015

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“…the human immune response, gene expression and cellular differentiation. One of the most important techniques used currently in single-molecule biophysics research is, unquestionably, fluorescence microscopy [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Single-molecule fluorescence microscopy review: shedding new light on old problems

2017

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Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

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“…In order that the physico-chemical characteristics of biomolecules can be experimentally determined, the majority of biophysics SME at present is carried out on molecules in vitro, isolated from confounding factors of their native environment. However, there have been recent successes involving SME in the true physiological context of the living cell, and such directions of research are likely to become significantly more widespread in the next few years [8].…”

Section: Introductionmentioning

confidence: 99%

Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time

Harriman

Leake

2011

J. Phys.: Condens. Matter

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The soft matter of biological systems consists of mesoscopic length scale building blocks, composed of a variety of different types of biological molecules. Most single biological molecules are so small that 1 billion would fit on the full-stop at the end of this sentence, but collectively they carry out the vital activities in living cells whose length scale is at least three orders of magnitude greater. Typically, the number of molecules involved in any given cellular process at any one time is relatively small, and so real physiological events may often be dominated by stochastics and fluctuation behaviour at levels comparable to thermal noise, and are generally heterogeneous in nature. This challenging combination of heterogeneity and stochasticity is best investigated experimentally at the level of single molecules, as opposed to more conventional bulk ensemble-average techniques. In recent years, the use of such molecular experimental approaches has become significantly more widespread in research laboratories around the world. In this review we discuss recent experimental approaches in biological physics which can be applied to investigate the living component of soft condensed matter to a precision of a single molecule.

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Using bespoke fluorescence microscopy to study the soft condensed matter of living cells at the single molecule level

Cited by 3 publications

References 11 publications

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time

Single-molecule fluorescence microscopy review: shedding new light on old problems

Single molecule experimentation in biological physics: exploring the living component of soft condensed matter one molecule at a time

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