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Application of Nonlinear Microscopy in Life Sciences
Published in Lingyan Shi, Robert R. Alfano, Deep Imaging in Tissue and Biomedical Materials, 2017
Zdenek Svindrych, Ammasi Periasamy
Superresolution techniques reach resolution that is higher than the Abbe limit (Eqs. 6.1 and 6.2). The main strategies to overcome the diffraction limit are: structured illumination (SIM) and saturated SIM, localization of single blinking molecules (PALM, STORM) or capturing intensity fluctuations of overlapping molecules (SOFI), and methods based on nonlinear behavior of fluorescence intensity (STED, RESOLFT). While all methods, that allow better than 2× improvement in resolution, rely on some nonlinear or random effect, all these methods may use multiphoton processes for excitation or activation of fluorescence. As examples, PALM with widefield temporal-focus 2P photoactivation in single plane [60] and with line-scan temporal focus 2P photoactivation [61] were demonstrated. Also, STED with 2P excitation using femtosecond pulses, and 1P depletion with stretched pulses of the same wavelength has been demonstrated [62].
Bridging the gap: Super-resolution microscopy of epithelial cell junctions
Published in Tissue Barriers, 2018
Emily I. Bartle, Tejeshwar C. Rao, Tara M. Urner, Alexa L. Mattheyses
Reversible saturable optical fluorescence transitions (RESOLFT) microscopy is a category of methods encompassing stimulated emission depletion microscopy (STED) and ground state depletion (GSD) microscopy. Like all super-resolution techniques, STED relies on measuring only a subset of fluorophores to overcome the diffraction limit.13,50,51 In most STED microscopes, two overlapping lasers are synchronized, one to excite the sample and the other to drive excited fluorophores to the ground state without emission of a photon, thereby depleting the fluorescence.52 Because of the “doughnut” shape of the depletion beam, only fluorophores in the center of the excitation emit fluorescence. The excitation and depletion beams are raster-scanned over the sample, as in a laser scanning confocal, to acquire a super-resolution image. An advantage of RESOLFT microscopy is that a super-resolution image is directly acquired, with no reconstruction necessary.
Super-resolution imaging and quantification of megakaryocytes and platelets
Published in Platelets, 2020
Abdullah O. Khan, Jeremy A. Pike
An extension of this approach is Reversible Saturable Optical Fluorescence Transitions (RESOLFT) microscopy, where the spatial manipulation of both organic fluorophores and photoswitchable fluorophores into their respective on-off states can achieve improvements in lateral resolution [27,28].