Real-time monitoring of infection processes requires the ability to link structural and functional dynamics across multiple spatial scales. Conventional wide-field fluorescence imaging has been effective in monitoring receptor recruitment and overall pathogen dynamics but remains challenged in resolving early binding events and the initial formation of microcolonies. Digital holography, while label-free, lacks the specificity to differentiate between surface-bound and internalized pathogens. Holotomography (HT), through quantitative 3D refractive index (RI) reconstruction, offers a structural modality that can be flexibly coupled with functional fluorescence approaches to address these gaps. We developed an integrated multimodal HT framework that combines refractive index tomography with complementary fluorescence modalities. At the single-cell scale, we implemented a correlative HT system with multicolor highly inclined laminated optical sheet (HILO) fluorescence microscopy to monitor Neisseria gonorrhoeae interactions with HeLa cells in vivo. This approach enabled volumetric tracking of bacterial binding alongside the spatiotemporal recruitment of CEACAM1 receptors, producing synchronized RI-fluorescence data cubes that revealed the dynamics of pathogen-host engagement. Extending this strategy to microbial communities, we integrated HT onto a water-immersion light sheet microscope optimized for E . coli biofilms. This platform couples 3D RI mapping of biofilm structure and extracellular polymeric substance heterogeneity with fluorescence correlation spectroscopy (FCS) readouts of local molecular diffusion. The combined RI-FCS measurements enabled direct correlation of biofilm refractive index profiles with molecular concentration gradients, providing structural-functional insights into the extracellular environment. Together, these multimodal platforms establish holotomography as a versatile structural imaging co-modality that can be integrated with diverse volumetric fluorescence techniques—from HILO for receptor dynamics to light-sheet FCS for biofilm transport studies. This integrated framework bridges structural and functional domains across single-cell and microcolony scales, offering a generalizable strategy for correlative infection imaging.
Yu et al. (Sun,) studied this question.