Figure 4.

Two-color excitation microscope (a) Microscope schematic, the fiber amplifier is overlapped in time (τ) relative to the diamond Raman laser using a mirror delay line. Their ratio of excitation power is adjusted using independent neutral density filters (ND). Both pulses are sent into a custom-built two-photon upright microscope. (b) 2C2P excitation demonstrated on a single cell expressing tdKatushka2-VASP-5 at various pulse synchronization delay times, scale bar is 10 μm. (c) 2C2P excitation as a function of delay time, τ, for various fluorescent labels. (d) The point spread function line profiles for 1055 nm, 1240 nm and 2C2P excitation on 200 nm red fluorescent beads in agarose (inset: image of a single 200 nm bead with 1055 and 1240 nm excitation overlaid in blue and red respectively, scale bar is 500 nm). DM1 is a shortpass dichroic filter with edge at 1180 nm. DM2 and DM3 are longpass dichroic filters with edges at 775 and 570 nm, respectively.