A video-rate laser scanning confocal microscopy system was implemented by adapting a previously developed confocal imaging platform.11,12Figure 1 depicts the schematic of the optical setup for imaging. Beam path of excitation beam and emitted fluorescence were marked by solid and dashed line, respectively. Three laser lines, constituted by dual-wavelength output DPSS laser emitting at 491 and 532 nm (Dual Calypso, Cobolt) and HeNe laser emitting at 635 nm (Radius, Coherent) were used as excitation lights for fluorescence imaging. To adjust power independently, dual-wavelength beams of 491 and 532 nm emitted from a DPSS laser were split to a single-wavelength beam by a dichroic beam splitter (DBS1; FF520, Semrock) and bandpass filters (BPF1; FF01-494/20, BPF2; FF01-531/22, Semrock). All three wavelength beams were combined by DBS2 (FF500/646, Semrock) and DBS3 (FF520, Semrock). Fast X-axis beam scanning at 17.28 kHz was achieved by rotating aluminum-coated polygon mirror (MC-5, Lincoln Laser). Combined with slow Y-axis beam scanning at 30 Hz achieved by a galvanometer-based scanner (6230H, Cambridge Technology), a two-dimensional raster beam scanning pattern was made and delivered to the back aperture of objective lens. Relaying lenses were selected to result in field of views (FOVs) of , and with , , and objective lens, respectively [UPlanFLN, numerical aperture (NA) 0.3; LUCPlanFl, NA 0.6; LUMFLN, water immersion, NA 1.1, Olympus]. The emitted fluorescence signals captured by the objective lens were de-scanned by back-propagating beam scanners. Then, multi-color fluorescence signals were separated from the excitation beams by DBS4 (FF494/540/650, Semrock) and split into three single color fluorescence signals by dichroic beam splitters (DBS5; FF555, Semrock, DBS6; FF650, Semrock) and bandpass filters (BPF3; FF01-512/25, BPF4; FF01-579/34, BPF5; FF01-676/29, Semrock). Three photomultiplier tubes (PMT; R9110, Hamamatsu) were used to detect the fluorescence signals, which were then digitized by an 8-bit 3-channel frame grabber (Solios, Matrox) at the speed of at ten million samples per second. Images were displayed at per frame at a frame rate of 30 Hz and stored in a hard disk in real time by using custom-written software based on Matrox Imaging Library (MIL9, Matrox).