Spectral analysis combined with advanced linear unmixing allows for histolocalization of phenolics in leaves of coffee trees

Abstract

An imaging method using spectral analysis combined with advanced linear unmixing was used to allow histolocalization of natural autofluorescent compounds such as hydroxycinnamic acid (chlorogenic acid) and xanthone (mangiferin) in living cells and tissues (mature coffee leaves). The tested method included three complementary steps: 1/ visualization of natural autofluorescence and spectrum acquisition with a multiphoton microscope; 2/ identification of some compounds using previous information on the chemical composition of the tissue, obtained from litterature; and 3/ localization of candidate compounds by spectral imaging. The second part of the study consisted of describing the histochemical structure of leaves during their development. This revealed very fast histochemical differentiation of leaves during the first week after their emergence. Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared. This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization. This also highlighted the paternal CAN origin of the leaf structure in the allotetraploid species ARA. The limits and advantages of the method without staining are discussed relative to classical epifluorescence microscopy under UV light. This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

Cytological and biophysical comparative analysis of cell structures at the microsporogenesis stage in sterile and fertile Allium species

“…In many cases, they are involved in biotic or abiotic interactions of plants with their environment (Bellow et al 2012). Thus, the fluorometric detection of these compounds can be used for the qualitative study of their role and dynamics in plant cells, as has been shown for coffee tree leaves, which were subjected to the spectral analysis focused on the histochemical differentiation of those leaves (Conéjéro et al 2014). …”

Mass Spectrometry Imaging of Specialized Metabolites for Predicting Lichen Fitness and Snail Foraging

“…For example, classical fluorescent techniques are based on the molecule chromophore, where metabolites are only differentiated with their emission wavelength (i.e. flavonoids and terpenoids at 525 nm approximately), so it appears to be a nonselective targeting [13][14][15]. More recently, Mass Spectrometry Imaging (MSI) has become a powerful tool to assess the spatial distribution of metabolites in organisms [16][17][18][19][20][21].…”

Functional Imaging in living Plants - Cell Biology meets Physiology

“…Their protocol involves twophoton excitation, spectral characterization of pure chemicals and advanced linear unmixing. Conejero et al (2014) are able to follow the amount and distribution of key phenolic compounds throughout the development of leaves of various Coffea species. By obviating the need for any staining, truly noninvasive histochemical analysis based on quantitative imaging is achieved.…”

“…New spectroscopic techniques for label-free imaging to investigate plant physiology are presented by Peter et al (2014) and Conejero et al (2014). Peter et al (2014) use spectro-microscopy and Statistical Analysis of Room Temperature Emission Spectra (SART) to characterize in vivo function of photosystems PSI and PSII in chloroplasts.…”

“…As this technique requires only moderate modification of a confocal microscope, it may be readily implemented by well-resourced laboratories. Conejero et al (2014) present a method combining spectral analysis with linear unmixing to facilitate histolocalization of phenolics in coffee leaves. Their protocol involves twophoton excitation, spectral characterization of pure chemicals and advanced linear unmixing.…”