Imaging of multi-color fluorescence emission from leaf tissues
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Multi-color fluorescence emission from leaf tissues is presented as a powerful reporter on plant biochemistry and physiology that can be applied both at macro- and micro-scales. The blue–green fluorescence emission is typically excited by ultraviolet (UV) excitation. However, this approach cannot be applied in investigating intact leaf interior because the UV photons are largely absorbed in the epidermis of the leaf surface. This methodological barrier is eliminated by replacing the UV photon excitation by excitation with two infra-red photons of the same total energy. We demonstrate this approach by using two-photon excitation for microscopy of Arabidopsis thaliana leaves infected by pathogenic bacterium Pseudomonas syringae. The leaf structures are visualized by red chlorophyll fluorescence emission reconstructed in 3-D images while the bacteria are detected by the green emission of engineered fluorescence protein.
KeywordsChlorophyll fluorescence Blue–green fluorescence Pyridine nucleotide Two-photon microscopy
Enhanced green fluorescent protein variant
Green fluorescent protein
Ultraviolet A radiation
This study was supported by “AUTOSCREEN for cell-based high-throughput and high-content gene function analysis and drug discovery screens” project of the Framework 6 program of the European Community (STREP 037897) and by the grant AV0Z60870520 of the Academy of Sciences of the Czech Republic.
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