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Super-Resolution Microscopy at a Glance

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Super-Resolution Microscopy at a Glance ARTICLE SERIES: Imaging Cell Science at a Glance 1607 Super-resolution visualization of ensemble perturbations. It is and >450–700 nm in the z direction. This limit, now possible to visualize the individual also called the point-spread function (PSF), is microscopy at a glance molecules as they dynamically interact. Super- the fixed size of the spread of a single point of resolution microscopy offers exciting light that is diffracted through a microscope; it is Catherine G. Galbraith1,* and opportunities for biologists to ask entirely new also a measure of the minimum-size point James A. Galbraith2,* levels of questions regarding the inner workings source or object that can be resolved by a 1National Institute of Health, 1NICHD and 2NINDS, of the cell. microscope. Objects that are smaller than the Bethesda, MD 20892, USA The impact of super-resolution microscopy is PSF appear to be the same size as the PSF in *Authors for correspondence ([email protected]; rapidly expanding as commercial super- the microscope, and objects that are closer [email protected]) resolution microscopes become available. together than the width of the PSF cannot be Journal of Cell Science 124, 1607-1611 However, super-resolution microscopes are not distinguished as separate. A commonly used © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.080085 based on a single technology, and the differences measure of the PSF width is the Rayleigh (R) between the individual technologies can criterion: R0.61l/NA, where NA is the Advances in microscopy and cell biology are influence how suited each approach is to address numerical aperture. Any microscopy technique intimately intertwined, with new visualization a specific cell biological question. Here, we that overcomes the resolution limit of possibilities often leading to dramatic leaps in highlight the main technologies and conventional light microscopy by at least a our understanding of how cells function. The demonstrate how the differences between them factor of two is considered to be a super- recent unprecedented technical innovation of can affect biological measurements. resolution technique. super-resolution microscopy has changed the Super-resolution techniques break the limits of optical resolution from ~250 nm to Defining super resolution diffraction limit by temporally or spatially ~10 nm. Biologists are no longer limited to The resolution limit of conventional light modulating the excitation or activation light. For inferring molecular interactions from the microscopy is ~250 nm in the x and y direction, example, structured illumination microscopy Journal of Cell Science (See poster insert) 1608 Journal of Cell Science 124 (10) (SIM) illuminates the entire field with a striped the PSF to reduce its effective diameter. They toactivation localization microscopy (PALM) pattern of light (Gustafsson, 2000). When this surround a laser-scanning focal excitation PSF (Betzig et al., 2006) and fluorescent PALM excitation pattern mixes with the spatial pattern with an annulus of longer-wavelength light that (fPALM) (Hess et al., 2006) use genetically of the sample they produce an interference has an intensity high enough to saturate all expressed photoactivatable probes to achieve pattern (called a moiré fringe) that is much fluorophores in the annulus to the ground state the on–off states of the fluorophore. An coarser than either pattern alone and is (STED) (Hell and Wichmann, 1994) or the alternative approach, stochastic optical detectable by the microscope. The excitation meta-stable dark state (GSD) (Bretschneider et reconstruction microscopy (STORM) (Rust et pattern is translated and rotated to generate a al., 2007) to suppress their fluorescent emission. al., 2006), uses pairs of cyanine (Cy) dyes, series of images with different moiré fringes. As Increasing the intensity of light in the annulus typically coupled to antibodies up to 15 nm in the illumination pattern is known, it can be expands the zone of saturation to create an length, to act as reporter and activator pairs in mathematically removed from the moiré to gain increasingly smaller excitation PSF that is order to cycle multiple times between the dark access to the normally irresolvable higher smaller in diameter than the diffraction limit of and light states. In direct STORM (dSTORM) resolution information in the sample. SIM 200 nm. The smaller PSF is then scanned over (Heilemann et al., 2008), several stand-alone increases resolution to ~100 nm in the x-y the sample to generate the enhanced-resolution synthetic dyes, such as Alexa-Fluor dyes, can direction and ~400 nm axially (Schermelleh et image. The best two-dimensional (2D) also be used in a blinking mode to attain super- al., 2008). SIM is limited to this factor-of-two resolution in a biological context for STED or resolution images. Despite the technical improvement because the periodicity of the GSD that has been achieved so far is 20–30 nm differences between these techniques, they all illumination pattern is created by diffraction- full width at half maximum (FWHM) across the localize the position of the molecule of interest limited optics and is, therefore, limited by the PSF (Westphal and Hell, 2005). The commercial to ~20 nm. PSF of conventional microscopy (Gustafsson, implementation of STED currently has a 2000). resolution of ~70 nm laterally with no increase The resolution of super resolution Other super-resolution imaging techniques in axial resolution above diffraction. Although The resolution of any image, conventional or modulate the excitation light to exploit the the excitation volume is below the diffraction super resolution, depends on the number of ability to saturate the emission of fluorophores limit, these techniques only make ensemble points that can be resolved on the structures in order to break the diffraction barrier by a measurements because they do not distinguish of interest. According to the Nyquist–Shannon greater amount. Saturation can be achieved by between individual molecules within an criteria (Shannon, 1949), a structural feature can using intense illumination to produce a excitation volume. only be resolved when the distance between two photophysical transition of the fluorophore to There are also good prospects for improving labels is less than half the feature size. a transient dark state that can lead to either a the resolution of SIM. The non-linear Therefore, at least two points need to be permanently dark state (bleaching) or the relationship between excitation and emission resolved within the minimum spatial feature size emission of light on a microsecond or can be combined with the illumination pattern that is to be imaged. Localizing individual millisecond time scale, which is much slower used in SIM. This technique – saturated SIM molecules with high precision does not create a than the nanosecond time scale of fluorescence. (SSIM) (Gustafsson, 2005) – can be thought of super-resolution image when there are not Alternatively, super-resolution techniques can as the inverse of STED, in which sharp dark enough labeled molecules within the PSF to Journal of Cell Science use light to induce photochemical reactions regions are created instead of sharp bright identify the spatial and temporal features of the in photoswitchable or photoactivatable regions. The resolution of SSIM scales with the structure. Thus, when a single-molecule-based fluorophores, and either transition them level of saturation, and 50 nm lateral resolution super-resolution image is reported as having a between on and off states or change their color. has been demonstrated using fluorescent beads resolution of 20 nm, the number must refer to As long as these transitions can be limited to a (Gustafsson, 2005). The general term for super- structural resolution and not simply indicate that subset of fluorophores that are spatially resolution techniques that make use of the image is only displaying molecules whose separated by the distance of the microscope PSF, the reversible non-linear switching of the centroids could be identified with 20 nm the molecules can be located with precision fluorophore state is reversible saturable optical certainty. approaching 5 nm. Super-resolution techniques fluorescence transitions (RESOLFT) (Hell, can be separated into two categories depending 2003). Speed of acquisition on whether these effects are exploited at the Many super-resolution techniques obtain ensemble level or at the single-molecule level. Single-molecule techniques increased resolution at the cost of the speed of Single-molecule-based techniques overcome image acquisition, simply because they use Ensemble techniques the diffraction limit by using light to turn on only conventional microscope optics and hardware. Ensemble-based techniques increase resolution a sparse subset of the fluorescent molecules of It takes longer to scan the smaller PSF of the by shaping the excitation light, and the interest. Even though the visualized molecules STED excitation beam across a specimen than it resolution of the images obtained by these that are turned on appear to have the size of a would take to scan the larger PSF of a techniques is determined by the size of the PSF in a conventional microscope, if they are conventional spot-scanning microscope. The super-resolution PSF. By contrast, single- separated from each other by at least 200 nm, temporal resolution of STED microscopy has molecule techniques rely on localization then the concept of single-molecule localization been reported as 35 mseconds per image with a precision, which is the uncertainty in the can be used to determine their centroids with 1.8 m ´ 2.5 m field of view, at a 2D identification of the center of a molecule’s PSF nanometer-level precision (Gelles et al., 1988; resolution of 62 nm (Westphal et al., 2008). and depends on photon output (Thompson et al., Yildiz et al., 2003). This process is repeated over However, with a larger field of view, imaging 2002). Importantly, localization precision is not many cycles, with new molecules turning on and speed slows down dramatically.
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