Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects

Lew, Matthew D.; Lee, Steven F.; Badieirostami, Majid; Moerner, W. E.

doi:10.1364/ol.36.000202

Cited by 121 publications

(86 citation statements)

References 18 publications

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“…Each of the aforementioned acronyms refers to the specific active control mechanism that is used to prevent single-molecule overlap in any one image by keeping the emitting concentration low, such as photoactivation, photoswitching, collisional flux from the surrounding solution, and pumping into reversible long-lived dark states. Three-dimensional localizations are possible with SMACM techniques by breaking the axial symmetry of the standard point spread function (PSF) (14)(15)(16), imaging in multiple focal planes simultaneously (17), or projecting the axial dimension onto a lateral dimension (18). These methods can achieve precisions below 100 nm in 3D over a 2-μm range (14,19,20).…”

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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.

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135

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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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

135

141

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“…The lower half of Figure 22 shows the application of the DH-PSF method to 3D surface imaging of Caulobacter labeled by the rhodamine spirolactam (172). Much remains to be done, as new point-spread function designs continue to appear (180)(181)(182), with the continuing goal to extract the maximum information from each tiny single-molecule emitter in the most efficient fashion.…”

Section: Figure 22 Here -mentioning

confidence: 99%

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

Moerner

2015

Rev. Mod. Phys.

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The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90's, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later superresolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized. The early days Introduction and early inspirationsI want to thank the Nobel Committee for Chemistry, the Royal Swedish Academy of Sciences, and the Nobel Foundation for selecting me for this prize recognizing the development of super-resolved fluorescence microscopy. I am truly honored to share the prize with my two esteemed colleagues, Stefan Hell and Eric Betzig. My primary contributions center on the first optical detection and spectroscopy of single molecules in the condensed phase(1), and on the observations of imaging, blinking and photocontrol not only for single molecules at low temperatures in solids, but also for useful variants of the green fluorescent protein at room temperature (2). This lecture describes the context of the events leading up to these advances as well as a portion of the subsequent developments both around the world and in my laboratory.In the mid-1980's, I derived much early inspiration from amazing advances that were occurring around the world where single nanoscale quantum systems were detected and explored for both scientific and technological reasons. Some of these were (i) the spectroscopy of single electrons or ions confined in vacuum electromagnetic traps (3-5), (ii) scanning tunneling microscopy (STM) (6) and atomic force microscopy (AFM) (7), and (iii) the study of ion currents in single membrane-embedded ion channels (8). But why not optical detection and ...

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“…[32 ], astigmatism was induced by a cylindrical lens as a shaping method, which resulted in a PSF whose ellipticity depended on axial position. Different refinements of this method have been proposed using more controllable shaping methods resulting in a spiral [64] or helical [35] PSF, which aim at increasing the range over which molecules can be localized and the isotropy of the localization precision. …”

Section: Current Opinion In Chemical Biologymentioning

confidence: 99%

A starter kit for point-localization super-resolution imaging

Manley

Gunzenhäuser

Olivier

2011

Current Opinion in Chemical Biology

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Super-resolution fluorescence imaging can be achieved through the localization of single molecules. By using suitable dyes, optical configurations, and software, it is possible to study a wide variety of biological systems. Here, we summarize the different approaches to labeling proteins. We review proven imaging modalities, and the features of freely available software. Finally, we give an overview of some biological applications. We conclude by synthesizing these different technical aspects into recommendations for standards that the field might apply to ensure quality of images and comparability of algorithms and dyes.

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Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects

Cited by 121 publications

References 18 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

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

A starter kit for point-localization super-resolution imaging

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