Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on ...Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent devel- opments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassi- nosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime （FLT）. Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy （FIDSAM）, has been developed. FIDSAM is capable of imaging low- expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of F6rster resonance energy transfer （FRET） for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quan- titative analysis of the photosystem I and II （PSI/PSII） ratio in the chloroplasts of living Arabidopsis plants at room tem- perature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiolog- ical and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.展开更多
Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image c...Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.展开更多
文摘Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techni- ques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent devel- opments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassi- nosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime （FLT）. Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy （FIDSAM）, has been developed. FIDSAM is capable of imaging low- expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of F6rster resonance energy transfer （FRET） for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quan- titative analysis of the photosystem I and II （PSI/PSII） ratio in the chloroplasts of living Arabidopsis plants at room tem- perature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiolog- ical and molecular topics can be addressed and valuable insights into molecular and subcellular processes can be obtained in living plants.
文摘Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.
