Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system

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799

We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10-to 20-nm precision over a large depth of field (2 m) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.microscopy ͉ photoactivation ͉ superresolution ͉ computational imaging ͉ PSF engineering F luorescence microscopy is ubiquitous in biological studies because light can noninvasively probe the interior of a cell with high signal-to-background and remarkable label specificity. Unfortunately, optical diffraction limits the transverse (x-y) resolution of a conventional fluorescence microscope to approximately /(2NA), where is the optical wavelength and NA is the numerical aperture of the objective lens (1). This limitation requires that point sources need to be Ͼ Ϸ200 nm apart in the visible wavelength region to be distinguished with modern high-quality fluorescence microscopes. Diffraction causes the image of a single-point emitter to appear as a blob (i.e., the point-spread function or PSF) with a width given by the diffraction limit. However, if the shape of the PSF is measured, then the center position of the blob can be determined with a far greater precision (termed superlocalization) that scales approximately as the diffraction limit divided by the square root of the number of photons collected, a fact noted as early as Heisenberg in the context of electron localization with photons (2) and later extended to point objects (3, 4) and single-molecule emitters (5-8). Because single-molecule emitters are only a few nanometers in size, they represent particularly useful point sources for imaging, and superlocalization of single molecules at room temperature has been pushed to the 1-nm regime (9) in transverse (2-dimensional) imaging. In the third (z) dimension, diffraction also limits resolution to Ϸ2n /NA 2 with n the index of refraction, corresponding to a depth of field of Ϸ500 nm in the visible wavelength region with modern microscopes. Improvements in 3D localization beyond this limit are also possible by using astigmatism (10, 11), defocusing (12), or simultaneous multiplane viewing (13).Until recently, superlocalization of individual molecules was unable to provide true resolution beyond the diffraction limit (superresolution) because the concentration of emi...

Section: Single-molecule Localization By Using the Dh-psfmentioning

confidence: 82%

Section: Single-molecule Localization By Using the Dh-psfmentioning

confidence: 99%

mentioning

confidence: 99%

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Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Pavani

Thompson

Biteen

et al. 2009

Self Cite

979

799

“…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%

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

Lew

Lee

Ptacin

et al. 2011

136

141

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

“…A superresolution 2D image of the sample can then be rendered by overlapping all single molecule localizations from all image frames. This principle of localization has also been applied to single particle tracking (10)(11)(12)(13)(14).Single molecule-based superresolution microscopy requires individual chromophores to be turned "on" and "off" sequentially, either by photoswitching or by other stochastic photophysical processes. Compared to synthetic photoswitchable probes, photoactivatable fluorescent proteins (PA-FPs) (15-20) are superior in many regards: PA-FPs can be genetically fused to target proteins and endogenously expressed in cells and organelles, allowing live cell imaging; moreover, the genetic expression of the PA-FP ensures that it has nearly 100% specificity and efficiency of labeling target protein(s), a level impossible to reach by staining and conjugative probes; finally PA-FPs are much smaller than dye-labeled antibodies, permitting higher labeling density in biological samples, allowing for higher imaging resolution according to the Nyquist criterion (21, 22).…”

mentioning

confidence: 99%

Near-isotropic 3D optical nanoscopy with photon-limited chromophores

Tang

Akerboom

Vaziri

et al. 2010

Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.3D bioimaging | nanoimaging | bacterial imaging | neuron imaging | biophotonics F luorescence microscopy allows the observation of biological samples in a highly sensitive, selective, and relatively noninvasive fashion. Moreover, fluorescence imaging tools can probe deeply into tissues to obtain volume images without physically isolating different sections. However, traditional fluorescence microscopy suffers from diffraction-limited resolution, far larger than actual molecular sizes. Recent development of two different superresolution (SR) fluorescence imaging methods has broken the diffraction limit and enables direct optical observation of biological features near molecular scales. One approach is to create a subdiffraction excitation pattern by stimulated emission depletion (1, 2) or structured illumination (3, 4), and the other approach is based on single molecule localization (5-9). An essential part of the single molecule localization methods involves centroid fitting of serial single molecule images at the detection plane in order to determine the lateral localization of each fluorescent molecule with precision limited not by diffraction, but mainly by its number of emitted photons. A superresolution 2D image of the sample can then be rendered by overlapping all single molecule localizations from all image frames. This principle of localization has also been applied to single particle tracking (10)(11)(12)(13)(14).Single molecule-based superresolution microscopy requires individual chromophores to be turned "on" and "off" sequentially, either by photoswitching or by other stochastic photophysical processes. Compared to synthetic photoswitchable probes, photoactivatable fluorescent proteins (PA-FPs) (15-20) are superior in many regards: PA-FPs can be genetically fused to target proteins and endogenously...