A new software program can map the movements of multiple particles within cells simultaneously, providing insight into cellular functions that are difficult — and sometimes impossible — to investigate using single-cell tracking methods. The software, developed by researchers at the University of Bonn and Wageningen University and Research, speeds the high-throughput process used to observe molecules in cells, enabling fivefold shorter measurement times than single-particle tracking, according to the researchers. In single-particle tracking, the molecule is marked with fluorescent light, and hundreds of photos per second are taken using a high-resolution microscope. By looking at the gaps between molecules and the distances traveled by a single molecule from one photograph to another, the researcher can tell whether the particles are moving freely inside the cell or interacting with other molecules. Biomolecules move faster than cameras can capture, leading to gaps in the film. If a single particle is being tracked, its missing movements can largely be filled in. Researcher Koen Martens, from the Institute for Microbiology and Biotechnology at the University of Bonn, works at the custom-built superresolution fluorescence microscope that he uses for his investigations. Courtesy of Volker Lannert/University of Bonn. “However, if two or more identical looking particles ‘glitch,’ it is often impossible to determine which particle on the first frame corresponds to which on the next frame,” said Koen Martens, postdoctoral researcher at the University of Bonn. When the paths of two particles cross or the particles are too close together, their movements cannot be identified using single-particle tracking, Martens said. Therefore, the molecules must be studied one after the other, in a time-consuming process that makes it difficult to observe the molecular activity. The software developed by Martens and his colleagues, called TARDIS, which stands for Temporal Analysis of Relative DIStances, resolves this problem. With TARDIS, an all-to-all distance analysis between localizations (that is, between the positions of the molecule in the individual photographs) is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles. The software uses probability calculations to compute all possible paths of the particles and determines which particles on each frame correspond to the particles on the subsequent frames. Instead of focusing on individual points, TARDIS looks at the entire sequence of movements within the cell and examines all the molecules simultaneously. “TARDIS makes the measurement process at least five times faster without any loss of information,” Martens said. The researchers tested the software on well-known molecular movement patterns such as diffusion. TARDIS performed accurately in complex conditions characterized by high particle density, strong emitter blinking, or false-positive localizations. It outperformed tracking algorithms when benchmarked on simulated and experimental data of varying complexity. “Our program calculated the correct movement,” Martens said. The computer does not require a lot of information for its calculations — just the coordinates of the particles at different time points, as measured with a microscope. TARDIS could help expand the possibilities for microbiological research. For example, it could be helpful in studying the effects of antibiotics and other medications on the molecular processes within cells. “Some antibiotics work by blocking specific molecular machines in the cell,” Martens said. By enabling the behavior of multiple molecular machines to be studied at the same time, TARDIS could provide insight into the effectiveness of an antibiotic and could do so rapidly. The technique is currently being used in research studying the process of DNA repair in single-celled organisms by observing the speed at which the repair function works. “Damage to our DNA activates molecules that repair it quickly, ideally before the cell divides and the damage spreads,” he said. “I don’t have a biological interpretation yet, but with my software, I can now, for the first time, follow the cell’s repair kit minute by minute.” The research was published in Nature Methods (www.doi.org/10.1038/s41592-023-02149-7).