Stimulated emission depletion (STED) microscopy has been established as an important technique for imaging below the diffraction limit facilitating new discoveries in an array of biological systems. In STED microscopy a “donut-shaped” laser focus is super-imposed onto the diffraction-limited focus of an excitation laser. The dounut-shaped beam suppresses fluorescence in the periphery of the excitation spot, reducing the effective point spread function to a sub-diffraction size. However, the application of multicolor STED microscopy in living cells poses a number of challenges. Here we detail a novel STED system specifically designed for two-color STED applications. Our system employs FPGA-based gated detection and fast beam scanning to reduce pixel dwell time and photobleaching. We demonstrate the instrument’s capability with two-color continuous imaging of intracellular targets below the diffraction limit allowing observation of rare events within live-cells.
Edward S. Allgeyer, Francesca Bottanelli, Emil B. Kromann, Xiang Hao, and Joerg Bewersdorf, "Development and application of 2-color live-cell STED nanoscopy
(Conference Presentation)," Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 97140D (Presented at SPIE BiOS: February 14, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2213728.4848767609001.
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