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Surface-enhanced infrared absorption (SEIRA) based on top-down fabricated nanostructures such as nanoantennas and metasurfaces has attracted much attention in recent years. These plasmonic resonant nanostructures can enhance the IR absorption signal of nearby molecules through its nearfield enhancement and have been shown to be able to detect adsorbed monolayers of proteins and lipids through their IR absorption spectra. Here, we demonstrate the continuous monitoring of cellular responses to stimuli using metasurface-enhanced infrared spectroscopy (MEIRS). A431 cells are seeded on a gold plasmonic metasurface fabricated on CaF2 substrate. Continuous monitoring is made possible by integrating the metasurface with a flow chamber, and the IR absorption spectra of the attached cells are measured in reflectance mode under continuous perfusion of cell culture medium. Scanning electron microscopy (SEM) revealed that the cells preferentially adhere to gold surfaces rather than CaF2 surfaces, suggesting that the IR signal measured through MEIRS is highly sensitive to the cells’ attachment and interaction with the gold metasurface. We have monitored the effect of methyl-beta-cyclodextrin, a cholesterol-depleting compound, on A431 cells. Principal component analysis highlighted the complex and subtle spectral changes of the cells. Keywords: MIR spectroscopy, surface-enhanced infrared absorption, metasurface, metasurface enhanced infrared absorption, MEIRS, cell adhesion, methyl-beta-cyclodextrin, cholesterol 

Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR–Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR–Cas systems. The type I-F CRISPR–Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR–Cas system.