A simple and practical method to acquire geometrically correct images with resonant scanning‐based line scanning in a custom‐built video‐rate laser scanning microscope

Summary Most currently available confocal or two‐photon laser scanning microscopes (LSMs) allow acquisition rates of the order of 1–5 images s −1 , which is too slow to fully resolve dynamic changes in intracellular messenger concentration in living cells or tissues. Several technologies exist to obtain faster imaging rates, either in the video‐rate range (30 images s −1 ) or beyond, but the most versatile technology available today is based on resonant scanners for horizontal line scanning. These scanning devices have several advantages over designs based on acousto‐optical deflectors or Nipkow discs, but a drawback is that the scanning pattern is not a linear but rather a sinusoidal function of time. This puts additional constraints on the hardware necessary to read‐in the image data flow, one of which is the generation of a pixel clock that varies in frequency with the position of the pixel on the scanned line. We describe a practical solution to obtain a variable pixel clock add‐on that is easy to build and is easy to integrate into a custom‐built LSM based on resonant scanning technology. In addition, we discuss some important hardware and software design aspects that simplify the construction of a resonant scanning‐based LSM for high‐speed, high‐resolution imaging. Finally, we demonstrate that the microscope can be used to resolve calcium puffs triggered by photolytically increasing the intracellular concentration of inositol trisphosphate.