Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system

Pavani, Sri Rama Prasanna; Greengard, Adam; Piestun, Rafael

doi:10.1063/1.3158923

Cited by 60 publications

(56 citation statements)

References 13 publications

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“…1A) (20,31,32). The DH-PSF converts the normal fluorescence spot of a single molecule into two spots by processing the image with a specially designed phase mask on a spatial light modulator.…”

Section: Resultsmentioning

confidence: 99%

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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.

136

141

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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

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Section: Resultsmentioning

confidence: 99%

“…1A). The imaging system convolves the standard PSF of the microscope with the DH-PSF using a phase-only spatial light modulator (Boulder Nonlinear Systems XY phase series) and has been described previously in detail (20,32,54). For this study, we illuminated C. crescentus cells with 514-nm light (Coherent Innova 90 Ar þ laser).…”

Section: Methodsmentioning

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.

136

141

View full text Add to dashboard Cite

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“…6 are √Avg(CRB) for 1.2 µm range around focus. The CRB calculation was done by method explained in Ref [40]. for NA 1.35 100X with detector pixel size of 16 µm at the wavelength of 670 nm.…”

Section: Precision Analysis and Experimental Crb Calculationmentioning

confidence: 99%

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE)

Grover¹,

DeLuca²,

Quirin³

et al. 2012

Opt. Express

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Super-resolution imaging with photo-activatable or photoswitchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field. We demonstrate for the first time a method integrating a Fisher information efficient DH-PSF design, a surface relief optical phase mask, and an optimal 3D localization estimator. 3D super-resolution imaging using photo-switchable dyes reveals the 3D microtubule network in mammalian cells with localization precision approaching the information theoretical limit over a depth of 1.2 µm. Bewersdorf, "Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples," Nat. Methods 5(6), 527-529 (2008

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“…Widefield methods, such as point-spread function engineering [10,11], holographic [12], off-focus [13], astigmatic [14,15] and multifocal-plane [16][17][18] microscopy, provide high-resolution 3D position detection. Another approach is to use the interference of light reflected by the particles and a reference surface to determine the absolute particlesurface distance.…”

Section: Introductionmentioning

confidence: 99%

In situ contrast calibration to determine the height of individual diffusing nanoparticles in a tunable confinement

Fringes

Skaug

Knoll

2016

Journal of Applied Physics

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We study the behavior of charged spherical Au nanoparticles in a nanofluidic slit as a function of the separation of the symmetrically charged confining surfaces. A dedicated setup called the nano-fluidic confinement apparatus (NCA) allows us to parallelize the two confining surfaces and to continuously approach them down to direct contact. Interferometric scattering (iSCAT) detection is used to measure the particle contrast with 2 ms temporal resolution. We obtain the confinement gap distance from the interference signal of the glass and the oxide-covered silicon wafer surface with nanometer accuracy. We present a three parameter model that describes the optical signal of the particles as a function of particle height and gap distance. The model is verified using nanoparticles immobilized at the glass and the substrate surface. For freely diffusing particles, the envelope of the particle signal as a function of gap distance and the known particle height at tight confinement is used to calibrate the particle signal in-situ and obtain all free model parameters.Due to the periodic contrast variation for large gap distances we obtain a set of possible particle heights for a given contrast value. For a range of small gap distances this assignment is unique and the particle height can be measured directly with high accuracy. The high temporal resolution allows us to measure the height occupation probability which provides a direct link to the freeenergy landscape the particles are probing via the Boltzmann distribution. Accordingly by fitting the results to a physical model based on the linear superposition approximation (LSA), the physical parameters governing the particle-glass interaction are quantified.1 arXiv:1511.05336v1 [cond-mat.soft]

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Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system

Cited by 60 publications

References 13 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

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE)

In situ contrast calibration to determine the height of individual diffusing nanoparticles in a tunable confinement

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