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Review
. 2009 Oct;20(8):886-93.
doi: 10.1016/j.semcdb.2009.08.006. Epub 2009 Aug 19.

Fluorescence microscopy below the diffraction limit

George H Patterson  1
Affiliations
Review

Fluorescence microscopy below the diffraction limit

George H Patterson. Semin Cell Dev Biol. 2009 Oct.

Abstract

Fluorescence imaging with conventional microscopy has experienced numerous advances in almost every limiting factor from sensitivity to speed. But improved resolution beyond the fundamental limitation of light diffraction has been elusive until recent years. Now, techniques are available that surpass this barrier and improve resolution up to 10 times over that of conventional microscopy. This chapter provides an overview of these new "super-resolution" imaging methods.

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Figures

Figure 1
Figure 1. Diffraction limits the resolving power of light microscopy
(A) When light is transmitted through a slit, it propagates radially upon exit. In this example, only five point source secondary wavefronts are depicted as the light exits the slit. The waves interfere constructively and destructively to produce a diffraction pattern. (B) Passage of light through an objective lens cannot focus the light to an infinitely small point as a consequence of transit through a circular aperture and this xz image of a 40 nm fluorescent bead demonstrates the resulting three-dimensional point spread function. (C) If PSF is projected onto a two-dimensional image, an Airy pattern with a bright central region and surrounding rings is produced. (D) This is the surface plot of the Airy pattern in (C). (E) Two fluorescent molecules will produce two observable spots >100 times larger than the molecules themselves. Plot profiles through the spots indicate the intensity distributions. As the two spots are moved closer together their summed intensities make difficult determining their individual fluorescent signals.
Figure 2
Figure 2. Sub-diffraction limited fluorescence imaging techniques
(A-C) Stimulated emission depletion microscopy (STED) uses one light source near the excitation maximum of a fluorophore. The Airy pattern of the excitation pulse is shown in (A). (B) A red-shifted light source with a zero node in the center of the Airy pattern and a wavelength located within the fluorophore's emission spectrum depopulates a subpopulation of molecules in the excited state. (C) The remaining excited fluorophores in the center of the Airy pattern are allowed to fluoresce normally. (D-I) Structured illumination microscopy (SIM) utilizes excitation of the sample with a known pattern of light, which produces Moiré fringes in the emission. Moiré fringes shown here are produced when a pattern is superimposed with another pattern oriented at various angles. The image in (D) was multiplied with itself with relative orientations of 5° (E) and 10° (F). The image in G has was also multiplied by the image in (D) rotated to 5° (H) and 10° (I). (J-O) High-density molecular localization relies on imaging single fluorescent molecules within a PSF. If the molecules can be imaged individually (J-K), their locations can be determined at sub-pixel precision using fits of their PSF (M-N). Several techniques, such as photoactivation and photoswitching, have been developed to “turn on” only a subset of molecules in maintaining the density of the fluorescent molecules low enough to distinguish single molecules. (O) Their calculated positions can then be plotted on an image using the uncertainty associated with the position information. By repeatedly or continuously “turning on” molecules, calculating their positions, and plotting them on a final image, super-resolution maps of structures can be produced or high precision dense molecular distributions can be determined and provide more information than simply imaging all molecules at once (L).
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