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High Spatio-Temporal-Resolution Detection of Chlorophyll Fluorescence Dynamics from a Single Chloroplast with Confocal Imaging Fluorometer

Chlorophyll 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. Here we demonstrated a novel 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 half-life period of the slow decay in the Kautsky curve, that is not possible to analyze with wide-field techniques. A linear relationship is found between excitation Intensity and the temporal positions of peaks/valleys in the Kautsky curve. 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. Confocal 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.