Abstract
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.
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