SPEND-hSRS imaging of fumarate uncovers mitochondrial metabolic heterogeneity

  • bioRxiv. 2026 Apr 7:2026.04.03.716311. doi: 10.64898/2026.04.03.716311.
Dingcheng Sun  1  2 ,  Guangrui Ding  3  2 ,  Haonan Lin  3  2 ,  Guo Chen  3  2 ,  Chun-Chin Wang  4  5 ,  Seema Bachoo  4  5 ,  Sarah E Bohndiek  4  5 ,  Ji-Xin Cheng  1  3  2
Affiliations
  • 1. Department of Biomedical Engineering, Boston University, Boston, MA, USA, 02215.
  • 2. Photonics Center, Boston University, Boston, MA, USA, 02215.
  • 3. Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA, 02215.
  • 4. Department of Physics, University of Cambridge, JJ Thomson Avenue, Cambridge, CB3 0HE, UK.
  • 5. Cancer Research UK Cambridge Institute, Robinson Way, Cambridge, CB2 0RE, UK.
PMID: 41993415 DOI: 10.64898/2026.04.03.716311
Abstract

Mitochondria, acting as the energy powerhouse, biosynthetic center, and reductive equivalent hub of the cell, participate in cellular metabolic activities. However directly imaging mitochondrial chemical content and quantifying metabolic activity in living cells remain challenging. Here, by Self-PErmutation Noise2noise Denoiser enhanced Hyperspectral Stimulated Raman Scattering (SPEND-hSRS) microscopy, we demonstrate fingerprint-region metabolic imaging of fumarate, a key intermediate in the tricarboxylic acid (TCA) cycle, with sub-millimolar sensitivity. In chemotherapy-stressed bladder Cancer cells, fumarate imaging revealed two mitochondrial subpopulations with divergent TCA metabolic preferences quantified by ratio metric analysis. Pixel-wise least absolute shrinkage and selection operator (LASSO) spectral unmixing further reconstructs fumarate and lipid maps, uncovering localized fumarate enrichment in protrusions. Extending to CH-window hyperspectral SRS imaging, we uncover the interplay between mitochondria and lipid droplets (LDs) in protrusions, where fatty acid is found to be released from LDs, to fuel the TCA cycle. Together, our work establishes SPEND-hSRS as high-resolution platform for linking fumarate to mitochondrial heterogeneity. Our results provide new insights into how mitochondrial heterogeneity and interaction with LDs drive Cancer cell adaptation to stress.

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