Quantitative imaging
of chlorophyll fluorescence
from intact leaves

 We have developed an instrument to spatially map chlorophyll fluorescence from intact leaves. Our instrument extends the capabilities of previous instruments by (1) measuring Fo in addition to other steady-state fluorescence parameters Fm, Fm’, F’; (2) imaging both rapid fluorescence induction kinetics and electron transfer in the two-electron gate of photosystem II; (3) allowing for imaged areas ranging from 4 mm2 to 6 cm2; and (4) allowing leaves to be free, attached to the plant, or held in a cuvette that allows for environmental control and simultaneous measurement of gas exchange parameters. Illumination is provided by a combination of 1200 red and blue LEDs that deliver measuring pulses, an over-saturation pulse, or continuous actinic light. Measurement of fluorescence yield is achieved by detection of fluorescence during short (typically <100 msec), weak (<1% actinic) pulses of blue light. The instrument’s electronics currently support a JAI progressive scan CCD camera (659 x 494; 8-bit; 10 Hz) but allow for easy upgrade to more sensitive or higher speed cameras. All instrument parameters (timing, intensities, LED color ratio, shuttering times) can be varied independently to provide flexibility for different experimental protocols. Control of the instrument, image capture, manipulation, analysis, and presentation are executed through a program developed specifically for the task. We are using this instrument to study how herbivory by insects affects the photosynthetic competence of the remaining leaf tissues. Initial results indicate that apparent rates of electron transport in linden bean are reduced by 40% at >5 mm from the point of herbivory by corn earworm. The ability to reduce photosynthesis away from the initial region of damage suggests that the effects of herbivory on plant production may have been greatly underestimated in the past. We are in process of modifying the instrument to simultaneously map chlorophyll fluorescence and gene transcription by fluorescence from green-fluorescent protein.

Tim Miller and Antony Crofts , Department of Biochemistry, University of Illinois ; Kevin Oxborough , Department of Biological Sciences, University of Essex ; Jason Hamilton and Evan DeLucia , Department of Plant Biology, University of Illinois

We gratefully acknowledge the Competitive Research Initiative at the University of Illinois for providing the funding for this project.