Intrinsic OPTIR imaging and spectroscopy enable label-free monitoring of viral infection in living cells.

Resolving biochemical signatures associated with viral infection progression within individual living cells, without introducing fluorescent labels that demand genetic accessibility and may perturb the process under study, remains a challenge for conventional optical approaches. Optical photothermal infrared (OPTIR) spectroscopy addresses this by providing label-free vibrational contrast with molecular bond specificity in live cells, combining submicron spatial resolution with strong suppression of the water background in aqueous media. Here, OPTIR discrete wavenumber imaging and point spectroscopy were applied to follow baculovirus infection in live Spodoptera frugiperda (Sf) 9 cells carrying no fluorescent reporter. Cells were measured within a CaF₂ sandwich configuration at 3, 6, 12, and 24 h post-infection. Imaging at the amide II protein band (1545 cm⁻1) and a nucleic acid band (1086 cm⁻1) provided spatial discrimination between infected and uninfected cells at each time point, with balanced accuracy reaching 0.90, while point spectroscopy raised the pairwise balanced accuracy to 0.94 and confirmed the biochemical basis of the image contrast. Multiclass separation across time points reached an overall accuracy of 0.71. Single-cell heterogeneity was quantified by uniform manifold approximation and projection (UMAP), and cross-replicate reproducibility served as internal validation. These findings demonstrate that OPTIR enables label-free monitoring of viral infection in living cells using intrinsic biochemical signatures alone, providing a practical alternative to genetically encoded fluorescent reporters.