Chlorophyll fluorescence: a technological beacon for collaboration

By Robert Furbank, Chief Investigator.


What is Chlorophyll Fluorescence?

Chlorophyll fluorescence is one of the three roads that light takes after it is absorbed by chlorophyll molecules inside the leaf.
When light is absorbed by plants, it can be used for photosynthesis, dissipated as heat or re-emitted as light. This third path is called chlorophyll fluorescence and around 1% of the light absorbed by a leaf is re-emitted in a lower energy wavelength, located in the red part of the light spectrum.  This happens inside the chloroplast membranes in the plant’s light harvesting apparatus called photosystem 2.


What does chlorophyll fluorescence measurements tell us about photosynthesis?

Chlorophyll fluorescence can be used as a sensitive measurement of photosynthetic rate, photo-damage and how effectively a plant is protecting itself from excess light energy.  The amount of fluorescence is broadly inversely proportional to the use of incoming light for photochemistry and photosynthetic electron transport. However, while more fluorescence can mean less photosynthesis and vice versa, fluorescence can also decline or be “quenched” by damage or stress, photo-protection and loss of energy to heat and through other regulatory processes.

A breakthrough in the early 1980’s revolutionised our ability to use chlorophyll fluorescence as a research tool in photosynthesis. The researchers built an instrument that could give short pulses (<1S) of light around 3 to 4 fold higher in intensity than sunlight and measure chlorophyll fluorescence before and after the flash and after a period of darkness.
Copyright © Charles Tambiah (All rights reserved - Worldwide).

This technique called PAM fluorometry (short for pulse amplitude modulated), was commercialised and due to LED technology is now an affordable tool for plant biology.

It allows us to separate the component mechanisms “quenching” fluorescence and to determine the proportion of energy going directly to photosynthetic electron transport. It can be carried out with a simple fibre optic or clip on device on single leaves or using a camera based imaging system with LED panels. Thus, we now have a rapid way of measuring a “surrogate” for photosynthesis, electron transport rate (ETR), now becoming a popular technique in phenomics platforms, as well as in the hands of basic researchers.




Measuring fluorescence after a period in the dark also allows us to sensitively detect the damage from excess light excitation or photo-inhibition.

How is the Centre using Chlorophyll fluorescence technologies?

Chlorophyll fluorescence imaging is used at the ARC Centre of Excellence for Translational Photosynthesis in several research projects.
Furbank_MeasurementWe use it to look for photosynthetic trait variation in model plants such as Brachypodium and Arabidopsis on the Trayscan / PlantScreen phenotyping platforms at the ANU and CSIRO. This is a technology that permits us to image chlorophyll fluorescence, plant temperature and growth desired traits or characteristics on thousands of plants a day.

We also use it to screen for mutants in transgenic work aimed at altering electron transport in tobacco, with the aim of understanding and improving its photosynthetic efficiency.

Viriand BerniCSIROFluorescence is also being combined with other technologies such as gas exchange and hyperspectral measurements to understand critical components of the photosynthetic process. This is the case of Centre’s research on wheat at CSIRO and on rice at the International Rice Research Institute (IRRI).

Researchers in Brisbane, at the University of Queensland,  are using chlorophyll fluorescence as their leaf based photosynthetic phenotyping tool in combination with gas exchange to map genomic regions associated with photosynthetic traits in sorghum.

Measuring fluorescence on single leaves in the lab and field and in single plants indoors is well established but how do we scale this to remote sensing and canopy scale measurements?

Experiments are underway to develop canopy based fluorescence methods which could be used at IRRI on rice, the University of Queensland (UQ) on sorghum and at CSIRO / The Australian National University (ANU) on wheat.

There are two methods to measure canopy or landscape level chlorophyll fluorescence.  One is an imaging method, called SIF or sun induced fluorescence. This method relies on parts of the sun’s visible spectrum where there is little light due to absorbance by oxygen in the atmosphere (often called “oxygen” or “Frauhofer” lines).

Some of these “silent” bands in the solar spectrum are where chlorophyll fluorescence can be measured with a sensitive imaging spectrometer.  However, this method does not allow us to separate the quenching components of fluorescence.
Another useful new tool is a laser based system called LIFT (Laser Induced Fluorescence Transients) which fires rapid short (microsecond) flashes at the canopy and uses a telescope to look at the fluorescence returning to the sensor. This is under development in Canberra for wheat and rice.

In the year of light, while plants are green, the colour of interest should be red, a rich part of the spectrum for photosynthetic measurements across the CoE for Translational Photosynthesis.

Further reading: Murchie E and Lawson T. (2013) Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. Journal of Experimental Botany. Read full article