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Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Palayret M, Armes H, Basu S, Watson AT, Herbert A, Lando D, Etheridge TJ, Endesfelder U, Heilemann M, Laue E, Carr AM, Klenerman D, Lee SF - PLoS ONE (2015)

Bottom Line: We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet.In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation.This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.

ABSTRACT
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

No MeSH data available.


Related in: MedlinePlus

Structural imaging mode.Fixed S. pombe expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting (A), or after applying the vlsPALM thresholds defined in Fig 2B(B). The corresponding diffraction-limited image of the two cells is shown as an inset in (A). Close-ups of the white rectangles in (A-B) are shown in (C-D). The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in (C-D)).
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pone.0125438.g003: Structural imaging mode.Fixed S. pombe expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting (A), or after applying the vlsPALM thresholds defined in Fig 2B(B). The corresponding diffraction-limited image of the two cells is shown as an inset in (A). Close-ups of the white rectangles in (A-B) are shown in (C-D). The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in (C-D)).

Mentions: We imaged fixed fission yeast cells expressing the cytosolic protein Cdc22 fused to mEos2 at its C-terminus, to demonstrate the increase in contrast that the structural imaging mode allows and reveal a cellular organelle. The fission yeast Schizosaccharomyces pombe (S. pombe) is a powerful and highly tractable eukaryotic model organism, often used to study the cellular responses to DNA damage and the process of DNA replication. In response to nitrogen starvation or to osmotic stress, large vacuoles appear in S. pombe cells in order to restore the concentration of the cytosol [24]. We used Cdc22, a protein which is highly expressed and freely diffusive in the cytoplasm of the yeast, to image, in contrast, this organelle. Applying the vlsPALM thresholds in structural imaging mode, an increase in contrast is observed in the super-resolved image (Fig 3B and 3D): some vacuoles only appear after thresholding as out-of-focus PSFs from below or above the vacuoles are detected and plotted in the non-thresholded picture (Fig 3A and 3C).


Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Palayret M, Armes H, Basu S, Watson AT, Herbert A, Lando D, Etheridge TJ, Endesfelder U, Heilemann M, Laue E, Carr AM, Klenerman D, Lee SF - PLoS ONE (2015)

Structural imaging mode.Fixed S. pombe expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting (A), or after applying the vlsPALM thresholds defined in Fig 2B(B). The corresponding diffraction-limited image of the two cells is shown as an inset in (A). Close-ups of the white rectangles in (A-B) are shown in (C-D). The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in (C-D)).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401716&req=5

pone.0125438.g003: Structural imaging mode.Fixed S. pombe expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting (A), or after applying the vlsPALM thresholds defined in Fig 2B(B). The corresponding diffraction-limited image of the two cells is shown as an inset in (A). Close-ups of the white rectangles in (A-B) are shown in (C-D). The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in (C-D)).
Mentions: We imaged fixed fission yeast cells expressing the cytosolic protein Cdc22 fused to mEos2 at its C-terminus, to demonstrate the increase in contrast that the structural imaging mode allows and reveal a cellular organelle. The fission yeast Schizosaccharomyces pombe (S. pombe) is a powerful and highly tractable eukaryotic model organism, often used to study the cellular responses to DNA damage and the process of DNA replication. In response to nitrogen starvation or to osmotic stress, large vacuoles appear in S. pombe cells in order to restore the concentration of the cytosol [24]. We used Cdc22, a protein which is highly expressed and freely diffusive in the cytoplasm of the yeast, to image, in contrast, this organelle. Applying the vlsPALM thresholds in structural imaging mode, an increase in contrast is observed in the super-resolved image (Fig 3B and 3D): some vacuoles only appear after thresholding as out-of-focus PSFs from below or above the vacuoles are detected and plotted in the non-thresholded picture (Fig 3A and 3C).

Bottom Line: We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet.In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation.This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.

ABSTRACT
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

No MeSH data available.


Related in: MedlinePlus