Collaborative Research: Tools4Cells: Real-time 4D imaging of bacterial populations at single-cell resolution

In nature, bacteria either swim freely to search for food or attach to surfaces, forming biofilms. Biofilms are groups of bacteria that stick together via secreted polymers and can be found on many surfaces—from rocks in streams to medical devices. While sometimes beneficial, biofilms often cause problems in industrial and medical settings because they are hard to remove and resist antibiotics.

Understanding how bacteria interact with biofilms and switch between swimming/non-swimming states is crucial for controlling harmful biofilms. However, studying these interactions has been challenging due to the lack of tools that can observe both types of bacteria simultaneously. This project aims to develop HoloCon, a new imaging tool that combines three-dimensional holographic imaging and fluorescent microscopy, allowing scientists to see both free-swimming bacteria and biofilms in three dimensions and observe their interactions in real time at the single-cell level.

HoloCon will visualize and quantify both bacterial populations in one setup, explaining how resident biofilms repel free-swimming cells attempting to colonize and invade them—a common scenario in natural environments like the ocean. HoloCon will be widely useful to researchers observing cell dynamics at high spatial and temporal resolutions, and to others imaging cell or particle trajectories in complex three-dimensional environments such as tissues.

The Broader Impacts of the project include its intrinsic merit as the project could impact the work of microbiologists, cell biologists, and developmental biologists. Research tasks will be integrated with educational missions to train future scientists and engineers and inspire public interest through classes, research, and outreach activities in high schools.

The major goal of the proposal includes both the development of a new tool for cell biology studies and the scientific discoveries enabled by the new tool. Specifically, in Aim 1, an innovative imaging tool named HoloCon will be developed, which represents the first system capable of simultaneously capturing 3D images of rapidly swimming bacteria and monitoring the dynamic evolution of biofilm architecture at single-cell level.

HoloCon is composed of three integrated modules: a real-time 3D particle tracking module based on digital in-line holography (DIH) integrated with machine learning, a customized spinning disk confocal microscope module that receives information from DIH, and a fully integrated data analysis module ready for the research community to use. Aim 2 centers on addressing the key scientific questions of how resident biofilms repel free-swimming cells attempting to colonize and invade the biofilm.

New information revealed by HoloCon with be combined with bacterial genetics, biochemistry, and surface engineering tools to paint a comprehensive picture about the interactions between the sessile and planktonic bacterial populations at sub-micron spatial resolution and <10 ms temporal resolution. The central hypothesis is that the interaction between a swimming cell and an established biofilm depends on the swimming behavior of the invading cell, the topography of the resident biofilm, and the biochemical interactions between cells and the extracellular matrix. Revealing the resistance mechanism to biofilm will add significantly to our understanding on the competition between bacterial populations in nature, and on how ecological functions are tightly coupled with the cell biology of individual bacterium.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.