To date, tracking the real-time motions of single nanoscale objects in the live cell environments is still challenging due to the complexity of the biological surroundings as autofluorescence and excitation-induced scattering tend to render observation of nanoprobes difficult. Here, we present a new optical platform, time-resolved 3D multi-resolution microscope (TR-3DMRM), that integrates time-gated real-time 3D single-particle tracking (TG-RT3DSPT) with contextual two-photon fluorescence lifetime image (two-photon FLIM) taking full advantage of FLIM imaging. TR-3DMRM allows us to lock on to single nanoscale objects in noisy environment for minutes with high spatiotemporal precision (down to 10 nm and 10 μs in all three dimensions) and record single-photon arrival times of the micron scale 3D cellular context, enabling time-resolved spectroscopy to be performed during 3D single-particle tracking experiments. To demonstrate the capabilities of this instrument, artificial beads matrix and dye-stained cells were used as tracking environments where freely diffusing giant quantum dots (gQDs) were followed. By analyzing both 3D trajectories of the gQDs and contextual two-photon FLIM images of the environments, we can extract the interaction dynamics between the different interfaces and the freely diffusing nanoparticles. We anticipate that this new platform can unveil enzymatic process and intracellular dynamics which will ultimately lead to new insights in both enzyme engineering and therapeutic cellular delivery strategies.

Time-resolved 3D multi-resolution imaging: Design, construction, and applications

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