Rapid 3D movements can be studied by two complementary high-speed microscopy methods: digital holographic microscopy (DHM) 5, 9, 10, 11, 12 and multifocal imaging (MFI) 13, 14, 15, 16.ĭHM relies on the interference between two waves: a coherent reference wave and a wave resulting from the light scattered by the object. Although methods such as confocal and light-sheet microscopy allow precise 3D recordings, these techniques lack either the time or spatial resolution or are constrained to a small field-of-view (FOV) that is too narrow to follow fast biological processes in 3D. Many biological processes happen on the micrometer to millimeter scale within milliseconds: in one second, insects flap their wings 100–400 times 1, 2, microorganisms swim 0.5–50 body lengths 3, 4, 5, cilia and flagella beat for up to 100 times 6, 7, and the cytoplasm of plants streams over a distance of 100 µm 8. Organisms, cells, and subcellular compartments continuously undergo 3D movements. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable. The sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. We characterize fluid flow and flagellar beating of human and sea urchin sperm with a z-precision of 0.15 µm, in a volume of 240 × 260 × 21 µm, and at high speed (500 Hz). Here, we provide an open-source 3D reconstruction algorithm for multi-focal images that allows using MFI for fast, precise, label-free tracking spherical and filamentous structures in a large FOV and across a high depth. Multifocal imaging (MFI) allows high-speed 3D imaging but is limited by the compromise between high spatial resolution and large field-of-view (FOV), and the requirement for bright fluorescent labels. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Many biological processes happen on a nano- to millimeter scale and within milliseconds.

Wings 3d combine imagenes