Click-free imaging of trehalose trafficking in live mycobacteria via azido photothermal probe (app)

Real-time tracking of intracellular carbohydrates remains a grand challenge. While click chemistry enables bio-orthogonal tagging of small molecules with azido groups, subsequent fluorescence visualization is hindered by larger fluorophores, potentially altering intrinsic metabolism. Here, we introduce a click-free, dye-free method for dynamic tracking of trehalose within single mycobacteria via mid-infrared photothermal (MIP) imaging of azido-trehalose. Through spectroscopic MIP imaging, the metabolic pathways of trehalose and its interactions with intracellular proteins is demonstrated without click reaction and photobleaching, offering valuable insights into mycobacterial physiology. Our approach paves the way for further advancements in studying metabolic dynamics within living cells.