Live-cell imaging of NADPH production from specific pathways

Senlian Hong, Tao Chen, Ling Liu, Chen Cao, Fengxiang Lv, Joshua D. Rabinowitz, Yanyi Huang, Xing Chen

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


As anconversion of essential cofactor for lipid biosynthesis and antioxidant defense, reduced nicotinamide adenine dinucleotide phosphate (NADPH) is produced via various pathways, including the oxidative pentose phosphate pathway (oxPPP) and the malic enzyme 1 (ME1)-catalyzed conversion of malate to pyruvate. Live-cell detection of NADPH production routes remains challenging. Here, we report tracing hydrides into lipid droplets (THILD), a chemical imaging strategy for the detection of pathway-specific NADPH generation in live cells. This strategy exploits deuterium (2H)-labeled glucose ([2H]Glc) tracers that transfer deuterides to NADPH via specific pathways. The NADP2H, in turn, transfers deuterides to lipids, resulting in accumulation of C-2H bonds in lipid droplets, which can be visualized by bioorthogonal stimulated Raman scattering (SRS) microscopy. We used this concept to demonstrate the imaging of oxPPP-produced NADPH using the oxPPP-specific tracer, [3-2H]Glc. Furthermore, the “switch on” of NADPH production by ME1 in differentiating adipocytes was imaged by [4-2H]Glc. Finally, comparison of [3-2H]Glc and [4-2H]Glc THILD imaging of adipocytes showed that hypoxia induces suppression of ME1-mediated NADPH production and oxPPP-produced NADPH becomes the main source.

Original languageEnglish (US)
Pages (from-to)1642-1648
Number of pages7
JournalCCS Chemistry
Issue number6
StatePublished - Jun 2021

All Science Journal Classification (ASJC) codes

  • General Chemistry


  • Bioorthogonal Raman imaging
  • Deuterium tracing
  • Metabolic reprogramming
  • Pathway specificity
  • SRS microscopy


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