TY - JOUR T1 - Multifocal imaging for precise, label-free tracking of fast biological processes in 3D JF - bioRxiv DO - 10.1101/2020.05.16.099390 SP - 2020.05.16.099390 AU - Jan N. Hansen AU - An Gong AU - Dagmar Wachten AU - René Pascal AU - Alex Turpin AU - Jan F. Jikeli AU - U. Benjamin Kaupp AU - Luis Alvarez Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/05/16/2020.05.16.099390.abstract N2 - 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 suffers from the compromise between spatial resolution and field-of-view (FOV), requiring bright fluorescent labels and limiting its application. Here, we present a new approach for high-resolution, label-free, high-speed MFI, based on dark-field microscopy and operative over large volumes. We introduce a 3D reconstruction algorithm that increases resolution and depth of the sampled volume without compromising speed and FOV. This allowed us to characterize the flagellar beat of human sperm and surrounding fluid flow with a precision below the Abbe limit, in a large volume, and at high speed. Our MFI concept is cost-effective, can be easily built, and does not rely on object labeling, making it broadly applicable.Competing Interest StatementThe authors have declared no competing interest. ER -