Fig. 1
FaST-PLIM method for intravital contextual imaging of oxygen and T cell dynamics. a FaST-PLIM scanning design. In conventional two-photon (2p) PLIM, each pixel receives one series of excitation gates and each unitary decay measurement period is at least > 10 lifetimes (complete decay). In FaST-PLIM, each pixel receives only one excitation gate per scan and the unitary decay measurement period is substantially shorter than 10 lifetimes (‘incomplete decays’). b Design of individual pixel dwell time whereby the excitation gate (2p Pulse) is preceded by Pre-Pulse. Blue dots represent photon counts, the red line is fitted model. c Results of Monte Carlo modeling of the dependence of lifetime variability on the accumulated pixel time. “Conv. 500 μs” and “Conv. 100 μs” represent, respectively, ‘complete’ or partial decay conventional methods, i.e. without pre-pulse, and with the corresponding pixel dwell times. “2PreP 100 μs” represents a partial decay method with pre-pulse and a 100 μs pixel dwell time. Model probe lifetime was 50 μs. d Intravital FaST-PLIM of oxygen (PtP-C343 probe) in lung. The mouse was initially ventilated with atmospheric air (~ 21% O2) followed by 100% O2, and then atmospheric air once more. The black areas correspond to alveolar air spaces and indicate no signal from the probe. e Intravital FaST-PLIM of in vivo oxygen in mouse calvarial BM. The arrows indicate blood flow direction. Lifetime image was intensity-weighted. f Lifetime values along the A-A’ dashed line. g Spatial frequency distribution of BM pO2 before (Alive, orange line) and after specimen asphyxiation (Dead, blue line). h Oxygen and fluorescence dynamics in calvarial BM of healthy CD2-DsRed/CD11c-YFP mouse infused with TRITC-dextran (blood supply marker) and PtP-C343. “b” indicates bone areas in which no signal from the probe was recorded. i Example of pO2 experience of an individual T cell as it moves along its track. j Overall frequency of instantaneous T cell velocities and local pO2