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

## TL;DR

This paper introduces a new imaging technique using BCARS microscopy to study plant chemistry at high resolution and speed, overcoming limitations of traditional Raman methods.

## Contribution

The first application of BCARS microscopy to plant samples, enabling fast and detailed chemical imaging.

## Key findings

- BCARS microscopy allows high-resolution imaging of plant tissues with 10 ms/spectrum acquisition times.
- The method successfully identifies chemical components like waxes, pectin, and chlorophyll in leaf cross sections.
- It reveals photosystem-specific changes in chlorophyll and carotenoids in intact and degraded leaves.

## Abstract

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.