Subcellular structures span a range of length scales from micrometers to nanometers, Structures too small to be studied in detail. Furthermore, three-dimensional (3D) images may be generated, for example, byįocusing into the sample at different depths.ĭespite these advantages, conventional fluorescence microscopy is limited by its spatial resolution, leaving many biological With spectrally distinct fluorescent probes. The availability of a broad range of fluorophores enables the simultaneous imaging of multiple targets by multicolor labeling Fluorescent labeling techniques such as immunofluorescence, fluorescence in situ hybridization,Īnd genetically encoded fluorescent tags (e.g., green fluorescent protein ) make it possible to label and to image specificīiochemical components of a sample ( Giepmans et al. The noninvasive nature of lightĪllows biological specimens to be imaged with little perturbation, enabling researchers to observe dynamic processes as they Previous Section Next Section INTRODUCTIONįluorescence microscopy is a versatile technique widely used in molecular and cell biology. Improvement of an order of magnitude over conventional optical microscopy has been experimentally demonstrated. Imaging and requires relatively simple experimental apparatus its spatial resolution is theoretically unlimited, and a resolution This approach is generally applicable to biological Of cultured cells, multicolor STORM, and three-dimensional (3D) STORM. The articleĭiscusses photoswitchable fluorescent molecules, STORM microscope design and the imaging procedure, data analysis, imaging It uses optically switchableįluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. In this article, we describe stochastic optical reconstruction microscopy (STORM), a methodįor superresolution imaging based on the high accuracy localization of individual fluorophores.
When observed with light, however, structural features smaller than ∼0.2 µm are blurred and areĭifficult or impossible to resolve. Subcellular structures and molecular complexes essential for biological function exist on length scales from The relatively low spatial resolution of the optical microscope presents significant limitations for the observation of biological
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