Observing grazing behavior transitions in Cafeteria roenbergensis with video-rate two-photon microscopy.

Grazing behavior of free-living aquatic heterotrophic nanoflagellates (HNFs) on bacteria plays a central role in shaping microbial community structure and driving nutrient cycling. However, direct observation of these predator-prey interactions has been limited by the rapid motility of flagellates and the transient nature of their encounters. To overcome these challenges, this study presents a novel application of video-rate two-photon fluorescence microscopy for high-resolution, real-time imaging of fast-moving microorganisms. Using the HNF Cafeteria roenbergensis as a model system, we investigate dynamic grazing interactions between fluorescently stained bacteria and the flagellates detected via their intrinsic cellular autofluorescence. This two-photon microscope combined with real-time imaging capability enables continuous observation of the full grazing sequence: contact, capture, ingestion, and digestion, at single-cell resolution. Quantitative analyses across varying prey concentration reveal phase-specific durations and saturation behavior in grazing activities. Furthermore, real-time tracking uncovers a previously unobserved transition in grazing dynamics across two feeding behaviors of flagellates from starved to fed states in motile flagellates. This technique provides a powerful new tool to study rapid microbial interactions in situ and can be broadly applicable to diverse microbe-microbe systems. With the integration of targeted fluorescent molecular probes, this technique offers significant potential to elucidate mechanical and biochemical processes underlying microbial feeding and communication.