Two-beam two-channel two-photon flow cytometry setup (T3TC).

Confocal microscopy images of (a) PMBC stained with CFSE (green) and DeepRed (red), (b) green channel of KB cells stained with G5-FI-FA and G5-DR-FA, and (c) Red channel of KB cells stained with G5-FI-FA and G5-DR-FA. The details are given in Sec. 4.

(a) S channel (green) and L channel (red) traces from an ear blood vessel of a CD-1 mouse injected with $2.3\times {10}^9$ yellow-green fluorescent (Ex505/Em515) $2.0\text{-}\mu \mathrm{m}$ microspheres at time zero and $11\min$ after. The control traces are shown to the left of the orange dashed line. The orange and blue dashed lines corresponds to the first and second injection, respectively. Each spike in the S channel corresponds to the fluorescent burst from microspheres passing through the excitation region. (b) Blow up of the dual-channel raw data showing detail of individual fluorescent peaks. (c) Number of detected events at different time points before and after injection. Each time point is represented by number of peaks above the background threshold during a period of $107\mathrm{s}$ . (d) S channel (green) and L channel (red) traces from an ear blood vessel of a NU/NU CD-1 mouse injected with $3.6\times {10}^6$ DeepRed-labeled splenocytes at time zero and $25\min$ after. The control traces are shown to the left of the orange dashed line. The orange, blue, black, and magenta dashed lines correspond to the first injection, the second injection, $2\mathrm{h}$ after initial injection, and $1\text{day}$ after, respectively. Each spike in the L channel corresponds to the fluorescent burst from DeepRed-labeled splenocytes passing through the excitation region. (e) Magnified dual-channel raw data. (f) Number of detected events at different time points before and after injection. Each time point is represented by the number of peaks above the background threshold during a period of $214\mathrm{s}$ .

(a) S channel (green) and L channel (red) traces from a blood vessel in the ear of a NU/NU CD-1 mouse injected with $300\mu \mathrm{L}$ $\left(5\mu \mathrm{M}\right)$ DeepRed solution at time zero. The control traces are shown to the left of the orange dashed line. The orange and black dashed lines correspond to the initial injection and $1.5\mathrm{h}$ after initial injection, respectively. Each spike in the L channel corresponds to the fluorescent burst from DeepRed fluorophores in cells passing through the excitation region. (b) Magnified dual-channel raw data at the time of injection showing the background rise in the L channel after injection of free DeepRed dye solution. Individual fluorescent peaks above the background noise can also be observed in the L channel after the injection of the free DeepRed dye solution. (c) The background two-photon excited fluorescent signal in the L channel at different time points after the injection of DeepRed dye solution. The fluorescent peaks above threshold were not counted in the background signal. The red dashed line is the linear fit of the log value of two-photon fluorescence against time. (d) Number of detected events at different time points before and after injection. Each time point is represented by the number of peaks above the background threshold during a period of $214\text{seconds}$ . (e) The $20\text{-}\min$ peak frequency dynamics in the blood vessel of a NU/NU CD-1 mouse after the injection of $300\mu \mathrm{L}$ of DeepRed solution. The frequency was calculated as the number of peaks in the L channel with 214 (red) or $107\mathrm{s}$ (blue) time duration. Both axes are on a log scale. The coefficient of the linear fit (black dashed line) is the average of the coefficients from both of the decay curves.