Localization-Based Super-Resolution Light Microscopy
Kristin A. Gabor,1,2,3 Mudalige S. Gunewardene,1 David Santucci,4 and Samuel T. Hess1,3,* 1 Department of Physics and Astronomy, 120 Bennett Hall, University of Maine, Orono, ME 04469 2 Department of Molecular and Biomedical Sciences, 5735 Hitchner Hall, University of Maine, Orono, ME 04469 3 Graduate School of Biomedical Sciences, 263 ESRB/Barrows Hall, University of Maine, Orono, ME 04469 4 Volen Center for Complex Systems, 415 South St., Brandeis University, Waltham, MA 02454
*
sam.hess@umit.maine.edu
Introduction Fluorescence microscopy is an essential and flexible tool
for the study of biology, chemistry, and physics. It can provide information on a wide range of spatial and temporal scales. However, since the inception of light microscopy, diffraction has limited the size of the smallest details that could be imaged in any sample using light. Because much of biology occurs on molecular length scales, interest in circumventing the diffraction limit has been high for many years. Recently, several techniques have been introduced that can bend or break the diffraction limit. Localization-based methods introduced in 2006 have reached this goal and are now rapidly growing in popularity.
Principles of Localization-Based Super-Resolution Microscopy Localization versus resolution. Diffraction blurs the
details of objects imaged by a lens-based far-field microscope system. Objects closer together than R0 = 0.61λ/NA are unresolved according to the Rayleigh criterion, where NA is the numerical aperture, λ is the wavelength of detected light, and R0 is ~200–250 nm for green light imaged by a high-NA objective. In contrast, localization, which is the determination
of the position of an object using its image, has been achieved with precision of ~1 nm [1], limited in principle only by the number of detected photons. Localization-based super-resolution microscopy [2–4]
relies on both the imaging of single molecules and the stochastic activation of sparse subsets of such molecules. Tis is oſten achieved by optical control of molecular transitions between bright and dark states. When the number of emitting molecules is limited, and the distance between molecules is greater than R0, the molecular images are distinguishable (Figure 1). By activating a subset, imaging it, photobleaching or deactivating it, and repeating this process for many subsets of molecules, coordinates of thousands of molecules can be obtained. Molecular positions are measured by fitting the molecular image with a two-dimensional Gaussian function or with the experimental point spread function (PSF). For reliable localization, no more than a few molecules per frame can be localized within any diffraction-limited area. Tus, many (for example, 100–10000) image frames are required to achieve a high density of molecules in the final rendered image. Once a sufficient number of these molecules are acquired and localized, the molecular positions are plotted to generate an image. Te effective resolution of the image obtained depends
Figure 1: Principle of localization-based super-resolution microscopy. By limiting the number of fluorescent molecules visible at once, the images of the individual molecules become distinguishable. (A) Molecules are initially in an inactive (non-fluorescent) state. (B) Sparse subsets of molecules are converted into a fluorescent state by the activation beam (purple). When excited by the readout laser (green), they are imaged (C) until deactivated or photobleached (D). Molecules are localized by fitting the image with a two-dimensional Gaussian. Cycles of activation (B, E), readout and localization (C, F), and photobleaching (D, G) are repeated for many subsets of molecules. Rendered images with few (H) and large number (I) of localized molecules show buildup of structural detail as density increases. (J) Conventional image with diffraction-limited resolution.
12 doi:10.1017/S1551929511000435
www.microscopy-today.com • 2011 July
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