Fingerprinting organelles in living systems with optical Boxcar enhanced fluorescence-detected mid-infrared photothermal (OBF-MIP) microscopy

Chemical imaging at the single-organelle level is crucial to biological and medical research, but a significant gap remains. Fluorescence-detected mid-infrared photothermal (F-MIP) imaging combines fluorescence with vibrational imaging, using photothermal effects as a bridge, and offers a promising approach. However, severe photobleaching in previous sample-scan continuous wave fluorescence excitation geometry hinders hyperspectral imaging, making high-throughput organelle fingerprinting challenging. To overcome this, we advanced F-MIP with a laser-scanning optical Boxcar-enhanced strategy (OBF-MIP) to achieve organelle-specific chemical imaging. The fingerprinting of lysosomes in C. elegans revealed distinct spectral features originating from degraded small molecules. Overall, OBF-MIP provides new opportunities to investigate the physiological functions of organelles.