title: Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves
abstract: Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species
keyphrases: extended depth-of-focus imaging chlorophyll fluorescence intact leaves microscope-based systems spatial resolution calculated photosynthetic parameters plant species leaf topology map individual cells response charge-coupled device maximum fluorescence yield minimum fluorescence yield variable fluorescence numerical aperture primary quinone acceptor algorithms development extended-focus images reconstruction biophysical research technique