FILM: Mapping organellar metabolism by mid-infrared photothermal modulated fluorescence

Metabolism unfolds within specific organelles in eukaryotic cells. Lysosomes are highly metabolically active organelles, and their metabolic states dynamically influence signal transduction, cellular homeostasis, and organismal physiopathology. Despite the importance of lysosomal metabolism, a method for its in vivo measurement is currently lacking. Here, we report a Fluorescence-detected mid-Infrared photothermaL Microscope (FILM) implemented with optical boxcar demodulation, AI-assisted data denoising, and spectral deconvolution, to map metabolic activity and composition of individual lysosomes in living cells and organisms. Using this method, we uncovered lipolysis and proteolysis heterogeneity across lysosomes within the same cell, as well as early-onset lysosomal dysfunction during organismal aging. Additionally, we discovered organelle-level metabolic changes associated with diverse lysosomal storage diseases. This method holds the broad potential to profile metabolic fingerprints of individual organelles within their native context and quantitatively assess their dynamic changes under different physiological and pathological conditions, providing a high-resolution chemical cellular atlas.