Chemical Fingerprint Imaging In Planta with Broadband Coherent Anti-Stokes Raman Scattering Microscopy

Plants are inherently complex systems dynamically interacting at different size scale levels. Spontaneous Raman microscopy links the molecular with the cellular structural level; however, as Raman scattering is a low-probability phenomenon, pixel dwell times for biological applications are not compatible with high-resolution imaging. Due to absorption and autofluorescence interferences, Raman methods are often restricted to pigment-poor regions in plant samples. Here, we apply broadband coherent anti-Stokes Raman scattering (BCARS) microscopy─a nonlinear optical counterpart of spontaneous Raman microscopy─for the first time on plant samples. We show that it generates Raman-like vibrational signals but with much faster acquisition times (10 ms/spectrum), facilitating large-area imaging in high resolution. Using a new optimized unmixing procedure in conjunction with existing, robust preprocessing methods, we can extract the chemical and spatially rich information from leaf cross sections from the upper cuticle to the chlorophyll fluorescence-dominated palisade and spongy mesophyll region. The method selectively extracts chemical components from the cuticle (waxes), cell walls (pectin, cellulose), and mesophyll (chlorophyll, carotenoids, lipoproteins, starch) and depicts the accumulation of calcium oxalate crystals, flavonols, and anthocyanins in vacuoles. Photosystem-specific spectral changes of chlorophyll and carotenoid signals in intact and degraded leaves reveal a chloroplast adaptation to the light absorption gradient in a leaf. The signal-intense bands give rise to further enhancement mechanisms through electronic (pre)resonance (chlorophyll, anthocyanin), strongly coherently amplified supramolecular ensembles with copigments (anthocyanin), and π-electron-phonon coupling (carotenoids). OH-stretching signals reveal that calcium oxalate crystals have a mixed hydration state. The results outline that the system view imaging capabilities of BCARS microscopy make it a valuable tool in plant and agrochemical research.