Abstract
BackgroundChlorophyll fluorescence (CF) is a key indicator to study plant physiology or photosynthesis efficiency. Conventionally, CF is characterized by fluorometers, which only allows ensemble measurement through wide-field detection. For imaging fluorometers, the typical spatial and temporal resolutions are on the order of millimeter and second, far from enough to study cellular/sub-cellular CF dynamics. In addition, due to the lack of optical sectioning capability, conventional imaging fluorometers cannot identify CF from a single cell or even a single chloroplast.Results and discussionHere we demonstrated a fluorometer based on confocal imaging, that not only provides high contrast images, but also allows CF measurement with spatiotemporal resolution as high as micrometer and millisecond. CF transient (the Kautsky curve) from a single chloroplast is successfully obtained, with both the temporal dynamics and the intensity dependences corresponding well to the ensemble measurement from conventional studies. The significance of confocal imaging fluorometer is to identify the variation among individual chloroplasts, e.g. the temporal position of the P–S–M phases, and the half-life period of P–T decay in the Kautsky curve, that are not possible to analyze with wide-field techniques. A linear relationship is found between excitation intensity and the temporal positions of P–S–M peaks/valleys in the Kautsky curve. Based on the CF transients, the photosynthetic quantum efficiency is derived with spatial resolution down to a single chloroplast. In addition, an interesting 6-order increase in excitation intensity is found between wide-field and confocal fluorometers, whose pixel integration time and optical sectioning may account for this substantial difference.ConclusionConfocal imaging fluorometers provide micrometer and millisecond CF characterization, opening up unprecedented possibilities toward detailed spatiotemporal analysis of CF transients and its propagation dynamics, as well as photosynthesis efficiency analysis, on the scale of organelles, in a living plant.
Highlights
Chlorophyll fluorescence (CF) is a key indicator to study plant physiology or photosynthesis efficiency
Fluorescence dynamics from a single chloroplast Conventional fluorometers observe CF dynamics over a large area on a leaf, and here we demonstrate that the confocal imaging fluorometer allows us to obtain CF transients from a precisely chosen cell or even a single chloroplast
The bottom and top of the box are the first and third quartiles, while the ends of the whiskers represent the maximum and minimum values. This result confirms that the averaged Kautsky curves acquired by the confocal fluorometer are similar to the curves taken with conventional fluorometers, but Intensity dependent fluorescence transient As we have mentioned in Fig. 2, it is well known that the Kautsky curve changes with intensity
Summary
Chlorophyll fluorescence (CF) is a key indicator to study plant physiology or photosynthesis efficiency. CF is characterized by fluorometers, which only allows ensemble measurement through wide-field detection. The typical spatial and temporal resolutions are on the order of millimeter and second, far from enough to study cellular/sub-cellular CF dynamics. Conventional imaging fluorometers (e.g. PAM and P&P fluorometers) are based on wide-field detection, and are routinely adopted to study ensemble of CF transients from a large area of a leaf, significantly limiting its spatiotemporal resolution. To study stress propagation in a plant leaf [17], current imaging fluorometers only provide spatial resolution on the order of millimeter, with temporal resolution on the order of second. To unravel the more detailed propagation dynamics, the required spatial resolution should be at least on single cell or sub-cellular level, while the temporal resolution should be enhanced to millisecond scale
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