Tracing fluorescence tracking’s past, present, and future

The ability to track fluorescent particles allows scientists to visualize the movement of single molecules or proteins during biological events. This technique has been used to learn about many processes within cells, like mRNA transport and immune signaling. Lance Xu and Steve Pressé investigate the history, significance, and future outlook of single-molecule tracking.

The authors present major advances for tracking using fluorescence microscopy, delving into the advantages and pitfalls of different tracking methods, including conventional widefield offline tracking and real-time confocal tracking.

“We are hoping that a broader outlook on tracking, and its potential to unravel trajectories with localization precision below the diffraction limit of light, is laid out for the public,” Pressé said. “Of particular interest is the importance of highlighting the many ingredients relevant to accurate tracking and explaining what the state-of-the-art is today.”

Despite the progress made in fluorescence tracking, the field faces challenges. For example, tracking deep within cells creates optical aberrations that degrade the resultant tracks. These must be addressed to understand molecular dynamics further within cells and in crowded environments. There is also uncertainty in the field around what conclusions can be made from biological molecular tracks featuring large uncertainty bars.

The authors discuss recent efforts to enhance the spatiotemporal resolution and computational efficiency of tracking techniques. For example, parallel computing could improve the efficiency of fluorescence tracking, and AI could improve the robustness of this technique in complex environments.

“With a plethora of AI tools becoming available to track, we thought this would be the right time to highlight advantages of physics-based approaches and how AI may contribute to strengthen these approaches in the future,” Pressé said.

Source: “Perspective: An outlook on fluorescence tracking,” by Lance W. Q. Xu and Steve Pressé, Journal of Chemical Physics (2025). The article can be accessed at https://doi.org/10.1063/5.0283108 .