Non-destructive, Functional Metabolic Imaging of Plant and Microbial Systems

Functional metabolic imaging of plant and microbial systems requires a sensitive, high spatial resolution microscopy method that can image metabolically important compounds in situ. This will require new chemically sensitive imaging methods to be developed. In this SBIR/STTR, we will develop a new coherent Raman-based microscopy technique called Doppler Raman that will improve sensitivity over existing stimulated Raman microscopy techniques by up to three orders of magnitude or more and use this technique to perform functional metabolic imaging of plant and microbial systems non-destructively. In Phase I, label-free imaging of metabolites in crude tobacco cell lysates was demonstrated by Doppler Raman. Compared with a current Raman approach, Doppler Raman’s detection sensitivity was orders of magnitude higher, and the detection was immune to fluorescence in highly autofluorescent tobacco plant cell lysates. We detected the endogenous biomarker nicotine in crude plant lysates and observed another peak that is consistent with heme. Both of these markers are implicated with stress signaling in response to insect attack, drought, heat and nutrient deficiency, making them ideal markers for plant health. In Phase II the sensitivity of Doppler Raman will be improved using the approaches conceptualized in Phase I. A Doppler Raman microscope will be assembled and the biomarkers nicotine and ATP/ADP will be imaged in live tobacco plant cells. The future commercial application and public benefits are a technology that is the first able to detect several metabolites simultaneously in crude lysates or intact cells without the use of fluorescent, radioactive, or Raman signal-enhancing tags. This new imaging approach and instrumentation will naturally contribute to other fields, such as the medical diagnostics and pharmaceutical fields.