Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

• Published in: Nature communications Vol. 12; no. 1; p. 4574
• Main Authors: Hansen, Jan N., Gong, An, Wachten, Dagmar, Pascal, René, Turpin, Alex, Jikeli, Jan F., Kaupp, U. Benjamin, Alvarez, Luis
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28-07-2021; Nature Publishing Group; Nature Portfolio
• Subjects: 14; 14/63; 631/1647/245/2186; 631/1647/245/2221; 631/1647/245/2226; 631/57; Algorithms; Animals; Biological activity; Biological Phenomena; Chlorophyta; Confocal microscopy; Field of view; Flagella; Fluid dynamics; Fluid flow; Fluorescence; Humanities and Social Sciences; Humans; Image reconstruction; Imaging, Three-Dimensional - methods; Labels; Male; multidisciplinary; Science; Science (multidisciplinary); Sea Urchins; Spatial discrimination; Spatial resolution; Sperm; Sperm Tail; Spermatozoa - cytology; Tracking
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-24768-4

Many biological processes happen on a nano- to millimeter scale and within milliseconds. 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. 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. 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. 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). The sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable.
Multifocal imaging suffers from a number of limitations. Here the authors report an open-source 3D reconstruction algorithm to enable label-free tracking of spherical and filamentous structures which they use to characterise fluid flow and flagellar beating of human and sea urchin sperm.