A 3D reconstruction method, developed by researchers at the ImmunoSensation2 Cluster of Excellence at the University of Bonn, the University Hospital, and the Center for Advanced European Studies and Research (caesar), uses multifocal imaging (MFI) devices to provide precise, label-free tracking of rapid biological processes in 3D. The tool allows scientists to use MFI to reconstruct the movement of quickly moving biological processes in 3D while maintaining a large field of view (FOV) and high spatiotemporal resolution. Standard MFI devices allow high-speed 3D imaging, but they require bright fluorescent labels and are limited by the need to compromise between high resolution and large FOV. An MFI device, when placed into the light path between the microscope and camera, splits the light collected by the objective and projects multiple focal images of the sample onto distinct locations of the camera chip. This approach constrains the FOV and lowers the signal-to-noise ratio when the number of focal planes is increased. For these reasons, 3D tracking based on MFI requires fluorescent labels and is limited by lower speeds, smaller sampling volumes, or reduced precision. A new algorithm provides novel insight into the dynamics of flagellar beating and its connection to the swimming behavior of sperm. Courtesy of René Pascal. To work around these constraints, the researchers developed a 3D reconstruction algorithm for use with MFI that is based on inferring the z-position of an object based on its defocused image — using the method to characterize fluid flow and flagellar beating of human and sea urchin sperm. They were able to track and reconstruct the sperm with a z-precision of 0.15 µm, in a FOV of up to 240 × 260 µm, at a high speed (500 Hz), and across a large depth of up to 21 µm. According to the team, these results show a 3.5-fold increase of depth, a 22-fold increase of FOV, and a 2.5-fold increase of sampling speed compared to conventional MFI systems. Additionally, the researchers’ method supported a sampling volume that allowed them to follow trajectories of individual sperm while simultaneously recording their flagellar beat in 3D over a long period of time. The researchers also determined the 3D fluid flow around the beating sperm. The large FOV allowed the researchers to relate swimming trajectories and beat pattern of sperm without compromising precision or speed. The team said that its approach could also be used to determine the 3D flow maps that result from the beating of motile cilia in the airways and brain, which beat in a way similar to the sperm tail. The researchers said that their method for enhancing MFI is cost-effective and easy to implement, making it accessible to a broad scientific community. Use of the algorithm does not require special equipment — a commercially available adapter that is compatible with most microscopes can be used. The system provides the flexibility to customize the focal distance between image planes using different adapter lenses, and it can be adapted to object sizes ranging from nano- to millimeter-scale using different objectives. The research was published in Nature Communications (www.doi.org/10.1038/s41467-021-24768-4).