Sub-micromolar imaging of intrinsic chromophores by two-photon photothermal microscopy captures mitochondrial response to chemotherapy

Abstract

Intracellular chromophores (e.g., NADH and FAD) play a central role in regulation of cellular metabolism. Though autofluorescence has been extensively used for label-free mapping of chromophores inside a cell, its sensitivity and molecular specificity are constrained by the low quantum yield and the fluorescence spectral overlap. Here, we address these challenges by employing a photothermal approach to measure the optical absorption of chromophores rather than its autofluorescence. By combining near-infrared pump and visible probe beams, our two-photon photothermal (2PPT) microscope exploits localized thermal transients generated through two-photon absorption, enabling detection of chromophore-specific signatures beyond the reach of autofluorescence. We demonstrate sub-micromolar limit of detection for the metabolic coenzymes NADH and FAD of 0.87 uM and 0.99 uM, respectively. Such high sensitivity enables differentiating the influence of different mitochondria shapes on metabolism activity. Importantly, the fluorescence crosstalk-free 2PPT can identify the biomolecular source of contrast from cellular mitochondria in a label-free manner based on spectroscopy. 2PPT microscopy is utilized to study metabolic alterations of mitochondria in cancer under chemotherapy at the single organelle level.