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The ability to monitor gene expression within living plants is of importance in many applications ranging from plant biology research to biofuel development; however, no method currently exists without requiring sample extraction. Herein, we report a multimodal imaging method based on plasmonic nanoprobes for in vivo imaging and biosensing of microRNA biotargets within whole plant leaves. This method integrates three different but complementary techniques: surfaceenhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The multimodal method utilizes plasmonic nanostars, which not only provide large Raman signal enhancement, but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross-sections. For the sensing mechanism, inverse molecular sentinel (iMS) nanoprobes are used for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will allow the study of these biotargets with previously unmet spatial and temporal resolution.
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