The schematic of real-time confocal and two-photon switchable intravital microscopy. (Upper panel) A schematic of the real-time confocal and two-photon intravital microscopy. In confocal mode, the continuous lasers excite the sample, and the emitted light is detected by confocal PMT. In two-photon mode, a Ti:Sapphire (femtosecond) laser shots red and near-infrared light, and the laser goes through a chirped-pulse amplifier, a special component that is necessary to prevent the pulse from damaging the parts in the laser. Then light excites the focal region of the sample. The two-photon PMT, which is closer to the objective lens, detects the two-photon fluorescence and second-harmonic generation signal. (Lower panel) The beam scanner can be operated via two different systems. Option 1 is a beam scanner consisting of a polygonal scanner (for the fast axis) and a galvanometer (for the slow axis). Option 2 is a beam laser consisting of two galvanometers (one for the fast axis, the other for the slow axis). Two different combinations can be used in real-time IVM for higher frame rates. The light path moves from (i) through (ii) to (iii), guided by the fast axis. The guided light then scans an x-axis line on the scan field. The light path then moves to (iv) along the y-axis, directed by the slow axis. Next, the light scans a next x-axis line on the scan field. Galvo, galvanometer; PMT, photomultiplier tube.