Fourier light-field imaging of human organoids with a hybrid point-spread function.

Volumetric interrogation of the cellular morphology and dynamic processes of organoid systems with a high spatiotemporal resolution provides critical insights for understanding organogenesis, tissue homeostasis, and organ function. Fluorescence microscopy has emerged as one of the most vital and informative driving forces for probing the cellular complexity in organoid research. However, the underlying scanning mechanism of conventional imaging methods inevitably compromises the time resolution of volumetric acquisition, leading to increased photodamage and inability to capture fast cellular and tissue dynamic processes. Here, we report Fourier light-field microscopy using a hybrid point-spread function (hPSF-FLFM) for fast, volumetric, and high-resolution imaging of entire organoids. hPSF-FLFM transforms conventional 3D microscopy and enables exploration of less accessible spatiotemporally-challenging regimes for organoid research. To validate hPSF-FLFM, we demonstrate 3D imaging of rapid responses to extracellular physical cues such as osmotic and mechanical stresses on human induced pluripotent stem cells-derived colon organoids (hCOs). The system offers cellular (2-3 µm and 5-6 µm in x-y and z, respectively) and millisecond-scale spatiotemporal characterization of whole-organoid dynamic changes that span large imaging volumes (>900 µm × 900 µm × 200 µm in x, y, z, respectively). The hPSF-FLFM method provides a promising avenue to explore spatiotemporal-challenging cellular responses in a wide variety of organoid research.

3D super-resolution live-cell imaging with radial symmetry and Fourier light-field microscopy.

Live-cell imaging reveals the phenotypes and mechanisms of cellular function and their dysfunction that underscore cell physiology, development, and pathology. Here, we report a 3D super-resolution live-cell microscopy method by integrating radiality analysis and Fourier light-field microscopy (rad-FLFM). We demonstrated the method using various live-cell specimens, including actins in Hela cells, microtubules in mammary organoid cells, and peroxisomes in COS-7 cells. Compared with conventional wide-field microscopy, rad-FLFM realizes scanning-free, volumetric 3D live-cell imaging with sub-diffraction-limited resolution of ∼150 nm (x-y) and 300 nm (z), milliseconds volume acquisition time, six-fold extended depth of focus of ∼6 µm, and low photodamage. The method provides a promising avenue to explore spatiotemporal-challenging subcellular processes in a wide range of cell biological research.