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.
- Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42Google Scholar
- Berger S, Benediktyova Z, Matous K, Bonfig K, Müller MJ, Nedbal L, Roitsch T (2007) Visualization of dynamics of plant-pathogen interaction by novel combination of chlorophyll fluorescence imaging and statistical analysis: differential effects of virulent and avirulent strains of P. syringae and of oxylipins on A. thaliana. J Exp Bot 58:797–806CrossRefPubMedGoogle Scholar
- Nedbal L, Whitmarsh J (2004) Chlorophyll fluorescence imaging of leaves and fruits. In: Papageorgiu GC, Govindjee (eds) Chlorophyll a fluorescence: a signature of photosynthesis. Springer, NetherlandsGoogle Scholar
- Pawley JB (1995) Handbook of biological confocal microscopy. Plenum, New YorkGoogle Scholar
- Rizzo MA, Piston DW (2004) Fluorescent protein tracking and detection. In: Goldman RD, Spector DL (eds) Live cell imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Ross FWD (1995) Fluorescence microscopy, vol 2. Cambridge University Press, CambridgeGoogle Scholar
- Vácha F, Sarafis V, Benediktyová Z, Bumba L, Valenta J, Vácha M, Sheue CR, Nedbal L (2007) Identification of photosystem I and photosystem II enriched regions of thylakoid membrane by optical microimaging of cryo-fluorescence emission spectra and of variable fluorescence. Micron 38:170–175CrossRefPubMedGoogle Scholar
- Wilson M, Lindow SE (1994) Inoculum density dependent mortality and colonization of the phylosphere by Pseudomonas syringae. Appl Environ Microbiol 60:2232–2237Google Scholar