Jikeli, J. F. | Search bioRxiv

bioRxiv2020

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

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 a new open-source 3D reconstruction algorithm for multifocal 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 high z-precision of 0.15 m, in a large volume of 240 x 260 x 21 m, and at high speed (500 Hz). The large 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.

cell biology↗

bioRxiv2018

SpermQ - a simple analysis software to comprehensively study flagellar beating and sperm steering

Motile cilia, also called flagella, drive cell motility across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g. in the brain or lung. For sperm, the picture has emerged that the motile cilium, also called flagellum, is not only a motor, but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum of the sperm cell navigates the sperm through a complex environment like the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behaviour of sperm. It has been proposed that distinct signalling domains in the flagellum control the flagellar beat. A detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signalling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the flagellar beat, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signalling components in sperm and distinct flagellar beat patterns.

cell biology↗