Fig. 7: Quantitative analysis of diffusional motion using the birefringence-based single-particle 3D tracking.

a Schematic illustrations of the sample configurations for the measurement of random diffusion using a sealed chamber (left) and directed diffusion using convection flow in an open chamber (right). This experiment was conducted using a 173 µm-thick mica substrate. b Representative time-lapse images of the 60 nm fluorescence nanoparticles freely diffusing in the sealed chamber. Three particles in the field of view are indicated by green, cyan, and blue arrowheads. The images were captured at a 1 Hz repetition rate with 100 ms exposure time. c Localized 3D diffusion trajectories of the three fluorescent nanospheres obtained from the time-lapse images in (b). d Mean square displacement (MSD) versus time lag plots obtained from the diffusion trajectories shown in (c) and (e). The dashed line shows the theoretically calculated MSD versus time lag plot of the nanospheres in a medium with 1 mPa s viscosity using the diffusion constant of the fluorescent nanospheres estimated by Eq. 7. The solid line shows fitting to the directed diffusion model (Eq. 6). Four sets of samples were prepared and tested to ensure data reproducibility. e Localized 3D diffusion trajectory of a fluorescent nanosphere obtained using the open chamber.