Engineered microbes can produce valuable chemicals but often require extensive optimization to reach sufficient production yields. Current quantitation methods such as gas chromatography-mass spectrometry are based on ensemble measurements that ignore the genetic or phenotypic variation among cells. Here, we use hyperspectral stimulated Raman scattering (hsSRS) to directly image free fatty acid synthesis in metabolically engineered Escherichia coli and uncover substantial heterogeneity among and within colonies. Using optimized laser exposures, we perform longitudinal SRS imaging to link the dynamics of biofuel synthesis with cell growth. Further, we develop an analytical protocol to enable mass spectrometry-like chemical specificity estimates of fatty acids.
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