Quantitative mapping of dynamic 3D transport in growing cells via volumetric spatio-temporal image correlation spectroscopy (vSTICS)
Quantitatively mapping three-dimensional (3D) flow, diffusion, and particle density in crowded living cells remains challenging because most dynamic optical microscopy measurements are effectively planar and existing analysis methods struggle with dense, noisy volumetric data. We introduce volumetric spatio-temporal image correlation spectroscopy (vSTICS), a framework that recovers voxel-resolved flow, diffusion coefficients, and particle densities from 3D fluorescence time series. Growing Camellia japonica pollen tubes were imaged with field-synthesis lattice light-sheet microscopy, and localized 3D spatio-temporal correlation analysis was applied to overlapping volumetric samples to generate maps of velocity, diffusion, and density. Validation with synthetic flow-diffusion simulations showed accurate recovery of seeded transport parameters, including velocities near $3$ $μ$m s$^{-1}$ and diffusion near $10^{-3}$ $μ$m$^2$ s$^{-1}$. Fluorescent microsphere experiments verified particle number and point spread function readouts and measured diffusion coefficients of $0.3 \pm 0.1$ $μ$m$^2$ s$^{-1}$ in gel, consistent with imaging-FCS measurements of $0.5 \pm 0.2$ $μ$m$^2$ s$^{-1}$. Applied to mitochondria in pollen tubes, vSTICS resolved a bidirectional reverse-fountain pattern with slower anterograde transport ($0.1$-$1$ $μ$m s$^{-1}$) and faster retrograde motion peaking near $3$ $μ$m s$^{-1}$, plus a retrograde corridor about $2$ $μ$m wide. Density and diffusion maps indicated a denser, more advective core and higher peripheral diffusion. High-density sub-diffraction vesicle mapping produced similar velocity landscapes with about ten-fold higher particle densities. These results establish vSTICS as a practical method for quantitative 3D mapping of intracellular transport and refines the reverse-fountain model by revealing asymmetric, predominantly transverse circulation.