Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane

Badieirostami, Majid; Lew, Matthew D.; Thompson, Michael A.; Moerner, W. E.

doi:10.1063/1.3499652

Cited by 118 publications

(111 citation statements)

References 16 publications

Supporting

Mentioning

107

Contrasting

Order By: Relevance

“…Notably, the SPRAIPAINT imaging strategy is independent of the 3D imaging method used to localize single molecules. Here, we utilized the DH-PSF for its excellent and uniform 3D localization precision across its whole depth of field (20,51), but other 3D superresolution methods (14,17) can be applied for 3D SPRAIPAINT as well. The choice of 3D method is largely influenced by the desired localization precision and depth of field required to image the target structure labeled with a candidate fluorophore.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Lew

Lee

Ptacin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

136

141

View full text Add to dashboard Cite

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, livecell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 × 12 μm).cell biology | wide-field microscopy | cytoskeleton | fluorescence microscopy | bacteria

show abstract

Section: Discussionmentioning

confidence: 99%

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Lew

Lee

Ptacin

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

136

141

View full text Add to dashboard Cite

show abstract

“…Among these strategies for 3D localisation, the double-helix PSF along with an optimised localisation algorithm offers better axial localisation precision (≈20nm) over an extended depth range (over 2 μm) than the astigmatic PSF or biplane imaging (≈65 nm) [63,64].…”

Section: Stochastic Localisation Microscopy In 3dmentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

View full text Add to dashboard Cite

Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

show abstract

“…This makes it harder to express the resolution of a super-resolution image, even for a single z-slice. This drawback does not afflict the double-helix method of 3D localisation microscopy, since in that system the lateral and axial localisation precisions are translationally invariant over the depth of field (Badieirostami et al 2010). In the double helix method, the uncertainty of z-positions could be estimated via a Fisher information approach, or by comparison with tabulated calibration data.…”

Section: Z-resolution In 3d Localisation Microscopymentioning

confidence: 99%

Blind assessment of localisation microscope image resolution

et al. 2012

View full text Add to dashboard Cite

Background: This paper analyses the resolution achieved in localisation microscopy experiments. The resolution is an essential metric for the correct interpretation of super-resolution images, but it varies between specimens due to different localisation precisions and densities. Methods: By analysing localisation microscopy as a statistical method of Density Estimation, we present a method that produces a blind estimate of the resolution in a super-resolved image. This estimate is derived directly from the raw image data without the need for comparisons with known calibration specimens. It is corroborated with simulated and experimental data. Results and discussion: Localisation microscopy has a resolution limit equal to 2σ, where σ is the r.m.s. localisation precision, evaluated as an average Thompson precision, Cramer Rao bound, or otherwise. Further, for a limited-sampling case in which there is only one localisation per fluorophore, the expected resolution of an optimised super-resolution image is worsened to approximately 3σ, due to smoothing processes that are necessarily involved in visualising the specimen with limited data. This 2σ or 3σ resolution can be estimated for any localisation microscopy specimen, and this metric can corroborate or replace empirical estimates of resolution. Other quantifiable resolution losses arise from sparse labelling, fluorescent label size, and motion blur.

show abstract

Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane

Cited by 118 publications

References 16 publications

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus

Fluorescence nanoscopy. Methods and applications

Blind assessment of localisation microscope image resolution

Contact Info

Product

Resources

About