Abstract: Laser scanning confocal microscope (LSCM) is an indispensable tool for life science research because of its high resolution and imaging ability of optical slices. The slow imaging speed of the commonly used XY and XYZ scanning methods limits the tracking of fast phenomena in biological research. This article described an XT line scanning mode for real-time fast imaging and established a method for identifying the direction of line scanning. The line segments in different directions were drawn through the fluorescent spheres, so that one end of the line segment was close to the center of the fluorescent sphere and the other end was far away. The XT line scanning was performed on the line segment, and the scanning direction was judged by the distance between the fluorescence signal and the two ends. The operating methods of XT line scanning in Zeiss, Nikon and Olympus confocal microscopes were described in detail, and the differences of the line scanning direction were compared. This method to determine the line scanning direction is simple and easy to operate, and has universal applicability, which can provide guide for application for microscopic imaging practitioners and researchers.

Key words: laser scanning confocal microscope, line scan, scan direction, fluorescent spheres