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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

Variation of the PSF in three dimensions.A vls (green plane, A) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, A). A fluorophore emitting from outside the vls (red volume above and below the vls, A), is blurred on the image plane of the detector (red optical path, A). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume (B) of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in (C-E). For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.
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pone.0125438.g001: Variation of the PSF in three dimensions.A vls (green plane, A) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, A). A fluorophore emitting from outside the vls (red volume above and below the vls, A), is blurred on the image plane of the detector (red optical path, A). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume (B) of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in (C-E). For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.

Mentions: SMLM methods overcome the diffraction limit of light by stochastically separating the emission of single fluorophores in time and, one by one, fitting their PSF in order to localise them with a precision proportional to , where N is the number of photons collected [17]. Such methods typically achieve localisation precisions of ~10 nm or lower in fixed cells, an order of magnitude better than conventional diffraction-limited systems. The PSF of a point-emitter is the impulse response of the optical system (composed of the objective and tube lenses), and is manifested as a diffraction-blurred punctum in the image plane, which is typically detected by an EMCCD [18] / sCMOS [19] camera (Fig 1A, green path). In standard wide-field microscope setups, the PSF of a point emitter is well approximated with a 2D-Gaussian [20] in the conjugate plane of the emitter. An emitter originating close to the focal plane of the imaging system will consequently be observed on the camera as a diffraction-limited PSF, well approximated with a 2D-Gaussian. However, an out-of-focus point emitter—that is, a single fluorophore whose position lies outside the focal plane—will have its conjugate plane located in front of (or behind) the plane of the camera, and as a result its PSF will be observed larger on the camera (Fig 1A, red path).


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)

Variation of the PSF in three dimensions.A vls (green plane, A) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, A). A fluorophore emitting from outside the vls (red volume above and below the vls, A), is blurred on the image plane of the detector (red optical path, A). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume (B) of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in (C-E). For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401716&req=5

pone.0125438.g001: Variation of the PSF in three dimensions.A vls (green plane, A) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, A). A fluorophore emitting from outside the vls (red volume above and below the vls, A), is blurred on the image plane of the detector (red optical path, A). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume (B) of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in (C-E). For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.
Mentions: SMLM methods overcome the diffraction limit of light by stochastically separating the emission of single fluorophores in time and, one by one, fitting their PSF in order to localise them with a precision proportional to , where N is the number of photons collected [17]. Such methods typically achieve localisation precisions of ~10 nm or lower in fixed cells, an order of magnitude better than conventional diffraction-limited systems. The PSF of a point-emitter is the impulse response of the optical system (composed of the objective and tube lenses), and is manifested as a diffraction-blurred punctum in the image plane, which is typically detected by an EMCCD [18] / sCMOS [19] camera (Fig 1A, green path). In standard wide-field microscope setups, the PSF of a point emitter is well approximated with a 2D-Gaussian [20] in the conjugate plane of the emitter. An emitter originating close to the focal plane of the imaging system will consequently be observed on the camera as a diffraction-limited PSF, well approximated with a 2D-Gaussian. However, an out-of-focus point emitter—that is, a single fluorophore whose position lies outside the focal plane—will have its conjugate plane located in front of (or behind) the plane of the camera, and as a result its PSF will be observed larger on the camera (Fig 1A, red path).

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