3-Dimensional super-resolution by spectrally selective imaging

827

739

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.fluorescence microscopy ͉ interferometry ͉ PALM ͉ photoactivated localization microscopy ͉ single molecule imaging A fundamental question in biomedical research is how specific, nanometer-scale biomolecules are organized into multicomponent micron-scale structural and signaling ensembles that facilitate cell function. For example, microtubules are built of 8-nm tubulin subunits that incorporate on the ultrastructural level into polymers 25 nm in diameter and Ͼ10 m in length that serve as the building blocks of superstructures such as mitotic spindles and flagella. However, key challenges remain for determining cellular ultrastructure with high molecular specificity. Because cellular structures are organized on the nanoscale, nanometer resolution is required. Immunoelectron microscopy (EM)-based approaches provide the necessary resolution, but they lack robust molecular specificity because the large size of the antibodies hampers their penetration into dense structures and the specificity of the antibody can be compromised by cross-reactivity and epitope masking caused by the harsh fixation often used for high-resolution EM. Fluorescence microscopy coupled with fluorescent protein (FP) fusion technology enables imaging cellular structure with exquisite molecular specificity, but the resolution of 3D images is diffraction-limited to Ϸ200 nm in the lateral and Ϸ500 nm in the axial direction, limiting conventional fluorescence to the characterization of cellular superstructure. Some of the recent fluorescence-based superresolution microscopy techniques (1-5) demonstrated a resolution of Ͻ100 nm in the vertical direction; however, this is still insufficient to bridge the resolution gap between cellular ultrastructure and superstructure. To achieve near-ultrastructural 3D resolution even for the limited photon outputs of highmolecular-specificity FPs, we have developed a single-photon multiphase interferometric scheme and integrated it with a lateral photoactivated localization microscopy (PALM) (6

Section: Resultsmentioning

confidence: 99%

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Shtengel

Galbraith

et al. 2009

827

739

“…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)(6)(7)(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.…”

mentioning

confidence: 99%

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

Pavani

Thompson

Biteen

et al. 2009

959

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...

“…A related technique using a single excitation beam (22) was demonstrated recently by van Oijen et al (25) for pentacene molecules embedded in a p-terphenyl host crystal at cryogenic temperature relying on differences in absorption peaks among molecules and using wide-field imaging. Previously, we reported a related colocalization scheme of two different-color dyes using NSOM that forced two different-wavelength excitation lasers through the same near-field aperture (thus ensuring overlapping excitation volumes).…”

mentioning

confidence: 99%

Ultrahigh-resolution multicolor colocalization of single fluorescent probes

Lacoste

Michalet

Pinaud

et al. 2000

284

233

An optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described in this paper. It relies on the use of two unique families of fluorophores, namely energytransfer fluorescent beads (TransFluoSpheres) and semiconductor nanocrystal quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A multicolor sample-scanning confocal microscope was constructed that allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps with a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread function of the microscope. We present results of two-dimensional colocalization of TransFluoSpheres (40 nm in diameter) and of nanocrystals (3-10 nm in diameter) and demonstrate distance-measurement accuracy of better than 10 nm using conventional far-field optics. This ruler bridges the gap between fluorescence resonance energy transfer, near-and far-field imaging, spanning a range of a few nanometers to tens of micrometers.A fter the completion of the human genome project, the cataloging of all gene sequences, and the acquisition of high-resolution structures of proteins and RNAs, future biological investigations will focus on how the fundamental cellular building blocks interact with each other. Another important issue will be to determine their precise locations in space and time in an attempt to decode and lay out the cell machinery and circuitry. Indeed, many vital functions of the cell are performed by highly organized structures, modular cellular machines that are self-assembled from a large number of interacting macromolecules and translocated from one cell compartment to another. To unravel the organization and dynamics of these molecular machines in the cell, a tool is needed that can provide dynamic, in vivo, three-dimensional (3D) microscopic pictures with nanometer resolution of individual molecules interacting with each other.Fluorescence microscopy can provide exquisite sensitivity down to the single molecule level for in vitro experiments (1-3). Moreover, it recently has been shown that single fluorophores can be detected in the membrane of living cells with good signal-to-noise ratio (S͞N) (4-6). What is not clear yet is whether single molecule fluorescence microscopy can provide the required spatial and temporal resolution. Technical challenges still to be met are (i) the synthesis of spectrally resolvable, bright, and stable fluorophores that can be coupled in vivo to macromolecules, (ii) the development of an easy-to-use and affordable instrument that permits high-resolution localization of individual point-like sources in 3D, and (iii) the ability to perform such measurements at a rate that is compatible with that of biological events.Recently, significant advances have been made in improving the spatial resolution of optical microscopy beyond the classical diffraction limit of light. These include (...