Biological and Biomedical Applications of Optical Photothermal Infrared Spectroscopy (O-PTIR)

Optical photothermal infrared (O-PTIR) spectroscopy is rapidly transforming molecular imaging by combining the chemical specificity of infrared absorption with the high spatial resolution enabled by visible-light excitation. This review provides a comprehensive and forward-looking overview of O-PTIR, with a particular focus on its expanding applications in biological and biomedical research. We begin by outlining the development of O-PTIR from earlier photoacoustic and photothermal infrared methodologies, placing it within the broader field of vibrational spectroscopy and emphasizing its unique advantages including sub-micrometer spatial resolution, label-free detection, and compatibility with heterogeneous biological samples. The foundational principles of O-PTIR, such as the photothermal effect, instrumental configurations, and the integration of simultaneous IR and Raman measurements, are discussed to establish the basis for its analytical capabilities. Leveraging these strengths, O-PTIR enables high-resolution and chemically specific imaging of key biomolecules including lipids, proteins, nucleic acids, and metabolites across cells, tissues, and microbial systems. Current applications span diverse areas such as cellular metabolism, microbial phenotyping, cancer diagnostics, biomarker identification, and pharmaceutical analysis. Alongside these advances, we critically evaluate existing limitations, including challenges associated with sample preparation, instrumental complexity, signal interpretation, and standardization. Finally, we highlight emerging directions such as live-cell O-PTIR measurements, artificial intelligence (AI)-driven spectral analysis, and the development of hybrid modalities including FISH-O-PTIR. Together, these innovations reinforce O-PTIR's potential as a transformative technology for biological and biomedical sciences, poised to bridge fundamental molecular studies with future clinical and translational applications.