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

摘要

Chlorophyll fluorescence (CF) is a key indicator to study plant physiology orphotosynthesis efficiency. Conventionally, CF is characterized by fluorometers,which only allows ensemble measurement through wide-field detection. Forimaging fluorometers, the typical spatial and temporal resolutions are on theorder of millimeter and second, far from enough to study cellular/sub-cellularCF dynamics. In addition, due to the lack of optical sectioning capability,conventional imaging fluorometers cannot identify CF from a single cell or evena single chloroplast. Here we demonstrated a novel fluorometer based onconfocal imaging, that not only provides high contrast images, but also allowsCF measurement with spatiotemporal resolution as high as micrometer andmillisecond. CF transient (the Kautsky curve) from a single chloroplast issuccessfully obtained, with both the temporal dynamics and the intensitydependences corresponding well to the ensemble measurement from conventionalstudies. The significance of confocal imaging fluorometer is to identify thevariation among individual chloroplasts, e.g. the half-life period of the slowdecay in the Kautsky curve, that is not possible to analyze with wide-fieldtechniques. A linear relationship is found between excitation Intensity and thetemporal positions of peaks/valleys in the Kautsky curve. In addition, aninteresting 6-order increase in excitation intensity is found betweenwide-field and confocal fluorometers, whose pixel integration time and opticalsectioning may account for this substantial difference. Confocal imagingfluorometers provide micrometer and millisecond CF characterization, opening upunprecedented possibilities toward detailed spatiotemporal analysis of CFtransients and its propagation dynamics, as well as photosynthesis efficiencyanalysis, on the scale of organelles, in a living plant.