Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.
Bibliographical noteFunding Information:
We thank Harald Kneipp for valuable discussions and support in setting up experiments. We thank D. Lajkó and V. Tarabykin (Charité-Universitätsmedizin Berlin; Anatomy, Institute of Cell Biology and Neurobiology) for providing access to the vibratome and Peter Lasch for providing CytoSpec v.2.00.01. We thank Oshry Markovich (The Hebrew University of Jerusalem, Israel) for advice in cultivating plants. Financial support by ERC Starting Grant no. 259432 (MULTIBIOPHOT) to J.K., by DFG (GSC 1013 SALSA) to Z.H. and by Einstein Stiftung Berlin (grant A-2011-77, I.Z., J.K., R.E.) is gratefully acknowledged.
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- 2-photon autofluorescence
- multivariate analysis
- second harmonic imaging
- spontaneous Raman