Fig. 2. LiFT-FRAP for noninvasive fast 3D diffusion tensor measurement.

a Schematic of the LiFT-FRAP system. A light sheet illuminates a thin slice of the sample and scans a 3D volume [inset (i)]. Emitted fluorescence is collected by the detection objective [inset (ii)]. A high-intensity bleaching laser creates a bleaching volume by performing 3D point-scanning at the center of the 3D illuminated volume [inset (iii)]. λ/2, half waveplate; PBS, polarizing beamsplitter; S, shutter; 2D GS, 2D galvanometer system; MM, MEMS mirror; IO, illumination objective; DO, detection objective; P, piezo stage; DIC, dichroic mirror; LPF, low-pass filter; T, tube lens; λ_ill, illumination laser; λ_det, detected emission fluorescence; λ_ble, bleaching laser; b LiFT-FRAP data collection and analysis workflow. In a LiFT-FRAP experiment, prebleaching images are first recorded, followed by the photobleaching process. Postbleaching images are collected instantly after bleaching. Time series of 3D LiFT-FRAP image data that record the 3D fluorescence recovery process was processed and then converted to the frequency domain through a 3D spatial Fourier transformation. Based on our 3D FRAP theory (Supplementary Note 5), the normalized solute concentration in the frequency domain $\tilde{C}/{\tilde{C}}_0$ (gray circles) will gradually decrease with the 3D fluorescence recovery (u, v, w are the spatial frequency coordinate. The unit of u, v, w is µm⁻¹). Fitted with the theoretical equations (red line), the diffusivity value is determined. Then each component of the 3D diffusion tensor can be calculated (Supplementary Note 5).