This approach relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. Resolutions approaching 20 and 50 nanometers in the lateral dimensions have been achieved with STED and SSIM, respectively.Ī second (and increasingly popular) strategy for overcoming the diffraction barrier employs photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution. By modifying the excitation light pattern through controlled engineering of the point-spread function to produce a much smaller focal spot size, these advanced techniques are capable of resolving fine structural details in biological specimens. Examples of this type of superresolution imaging are stimulated emission depletion ( STED), ground state depletion ( GSD), and saturated structured illumination ( SSIM) microscopy.
One highly developed strategy, often referred to as point-spread function engineering or illumination-based superresolution, utilizes non-linear optical approaches to reduce the focal spot size. In the past decade, several distinct conceptual strategies have been introduced to overcome the diffraction barrier and enable the analysis of biological structures at the superresolution level. Due to the fact that most subcellular structures (such as actin fibers, intermediate filaments, microtubules, ribosomes, and transport vesicles) exhibit features much smaller than this size, a mechanism for breaking through the diffraction barrier, and imaging beneath the size limitation that it defines, has been the holy grail of optical microscopy for centuries. The wavelike character of diffracted light prevents objects smaller than approximately 200 nanometers in the lateral ( x, y) dimensions and approximately 500 nanometers in the axial ( z) dimension from being visualized as anything but a blur. The widespread availability of advanced new instrumentation and highly sensitive detector systems has further permitted the acquisition of superior images with spatiotemporal characteristics appropriate for addressing a diverse array of biological questions.ĭespite its revolutionary impact on biology, all traditional forms of fluorescence microscopy (including widefield, confocal, and multiphoton) face a resolution limit imposed by the diffraction of light through lenses and circular apertures. Central to the recent dramatic rise in the use of fluorescence microscopy in cell biology has been the development of genetically-encoded fluorescent proteins that act as endogenous labels to enable virtually any protein or peptide to become a fluorescent homing beacon for imaging and analysis. Using these techniques, key insights into numerous events occurring within cells, tissues, and whole organisms have been obtained, including intracytoplasmic transport of vesicles, cytoskeletal structure, mechanisms of tissue remodeling, and the migration of cancer cells in a diseased organism.
Widefield and confocal fluorescence microscopy are capable of magnifying and imaging light-emitting fluorophores with a resolution that approaches a quarter of a micrometer, thus enabling the study of dynamic events and the fine structural details of cellular architecture.
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