2015
DOI: 10.1039/c5fd00077g
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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

Abstract: 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 buddi… Show more

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Cited by 76 publications
(117 citation statements)
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References 49 publications
(104 reference statements)
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“…We estimate that our calibration yields an axial resolution of ~100 nm, roughly 2-3 times poorer than our measured lateral resolution of ~40 nm under comparable imaging conditions [31]. This reduction in spatial resolution from lateral to axial is similar to other previously implemented 3D light microscopes [25] and compares favorably with other astigmatism based microscopes.…”
Section: Calibration and Performance Of The Microscopesupporting
confidence: 77%
“…We estimate that our calibration yields an axial resolution of ~100 nm, roughly 2-3 times poorer than our measured lateral resolution of ~40 nm under comparable imaging conditions [31]. This reduction in spatial resolution from lateral to axial is similar to other previously implemented 3D light microscopes [25] and compares favorably with other astigmatism based microscopes.…”
Section: Calibration and Performance Of The Microscopesupporting
confidence: 77%
“…3 A and Supplemental Movie S4). We monitored the spatiotemporal dynamics of foci in the planar membrane regions using automated tracking software (38), which allowed foci to be tracked for up to 18 s to a spatial precision of ∼40 nm (52) below the diffraction limit, thus enabling super-resolution localization data to be obtained. The measured width of the focal waist (defined as the half-width at half-maximum, determined from their pixel-intensity profile) was in the range of 200–300 nm, consistent with the PSF width of our microscope (Supplemental Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Such methods can enable tracking of single fluorescent proteins over very rapid millisecond time scales which significantly reduces motion blur of diffusing fluorescently labelled molecules in different compartments of live cells (Figure 2A), especially in the cytoplasm in which the viscosity is relatively low and so the rate of diffusion is relatively high [33]. These rapid imaging single-molecule fluorescence microscopy techniques may also be combined with convolution analysis of live cell fluorescence images to determine the copy number of proteins in a single cell, and indeed in separate cellular compartments [97]. Most super-resolution techniques, however, are mainly based on conventional fluorescence and confocal microscopy principles [98], but have resulted in huge advances in our knowledge of the biosciences, in particular concerning how processes operate in functional, living cells.…”
Section: Main Techniques and Applications Of Single-molecule Fluorescmentioning
confidence: 99%