Instead, the light wave forms a blurry focal spot with a finite size due to diffraction ( Figure 1Afig1). When light is focused by the objective of a microscope, the notion of light “rays” converging to an infinitely sharp “focal point” does not happen. In this Primer, we review the technological advances in the burgeoning field of “super-resolution fluorescence microscopy.” Then, we describe the application of these techniques to various areas of biology, which have quickly demonstrated the great promise of this exciting new area of bioimaging.īeating the Diffraction Limit of Resolution Most importantly, these techniques are beginning to provide insights into biological processes at the cellular and molecular scale that were hitherto unattainable. Recently, these research teams have developed several optical microscopy techniques that have shattered the diffraction barrier, improving spatial resolution by an order of magnitude or more over the diffraction limit. However, such limitations have not deterred a small group of scientists from pursuing “super-resolution” fluorescence microscopy that breaks through this seemingly impenetrable barrier. Because this property is intrinsic to all waves, breaking the diffraction barrier of light microscopy has been deemed impossible for a long time. The resolution for optical microscopy is limited by the diffraction, or the “spreading out,” of the light wave when it passes through a small aperture or is focused to a tiny spot. This resolution is approximately the size of an organelle and thus is inadequate for dissecting the inner architecture of many subcellular structures. However, the application of fluorescence microscopy to many areas of biology is still hindered by its moderate resolution of several hundred nanometers. Browse through any cell biological journal, and the impact of fluorescent microscopy is obvious, with > 80% of the images of cells in the book usually acquired with a fluorescent microscope. The unrivaled combination of molecule-specific contrast and live-cell imaging capability makes fluorescence microscopy the most popular imaging modality in cell biology. Even single molecules within a living cell can be visualized when these labeling strategies are combined with highly sensitive optical schemes and detectors ( Lord et al., 2010 Xie et al., 2008). For example, fluorescence in situ hybridization (FISH) detects DNA and RNA molecules with specific sequences, whereas immunofluorescence labels and fluorescent proteins allow the imaging of particular proteins in cells ( Giepmans et al., 2006). Indeed, one major element that makes light microscopy so powerful in biological research is the development of various staining methods that permit the labeling of specific molecules and cells. Then, ~200 years later, Ramón y Cajal used light microscopes to visualize Golgi-stained brain sections and create beautiful drawings of neurons, which led to his ingenious vision of how information flows in the nervous systems and helped to form modern neurobiology. For instance, more than 300 hundred years ago, Antonie van Leeuwenhoek used his self-ground optical lenses to discover bacteria and commence the field of microbiology. In fact, entire fields of biology have emerged from images acquired under light microscopes. For centuries, light microscopy has greatly facilitated our understanding of how cells function.
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