Chlorophyll Fluorescence

Chlorophyll fluorescence captured by Google Streetview camera cars with broken filtersArborcheck has been developed in response to the scale in which chlorophyll fluorescence is gaining popularity with Arborists around the world as an effective and comprehensive method of assessing tree health. The techniques, principles and instrumentation involved have been and continue to be widely used in photosynthesis research projects the world over.

Researchers such as Dr. Glynn Percival (Univ. of Reading, UK and Plant Physiologist and Technical Support Specialist for the F.A Bartlett Tree Expert Company Ltd) and Prof. Francesco Ferrini (Univ. of Florence, Italy) have been using chlorophyll fluorescence measurements in their work for many years and thanks to their stimulating and informative presentations, increasing numbers of people within the Arboricultural Consultation industry are becoming aware of chlorophyll fluorescence and how effective it can be in detecting tree stress long before any visible signs of decline.

But what actually is chlorophyll fluorescence?

Leaves can essentially be likened to photovoltaic panels in that their main purpose is to take energy from the sun and convert it to a different form of energy; in this case, complex carbohydrates that the tree may use for nutritional value. This is the main principle of photosynthesis.

No system is entirely perfect. The photosynthetic mechanisms within the leaf are unable to convert all the energy they receive from the sun into food. Consider this – on a hot sunny day, you or I would move into the shade if the sun’s rays became too intense. Obviously, a tree can’t do this so there has to be a method of dissipating the energy that simply can’t be used for photosynthesis. A leaf has 2 outlets for the solar energy it receives:

  1. Photochemical processes

    Photochemical processes channel the suns energy through the necessary steps required to drive photosynthesis. A good healthy tree is able to use about 80 – 84% of the sunlight received at the leaf surface for photosynthesis

  2. Non-photochemical processes

    Non-photochemical activities dissipate energy from the leaf in a manner that is not involved with driving photosynthesis. Dissipated energy is re-emitted from the leaf in the form of infra-red radiation (heat) or far-red radiation (chlorophyll fluorescence).

Let’s take an example – please refer to the diagram below. A period of drought causes a reduction in the rate of photosynthesis. The tree is able to convert a reduced amount of received solar energy by photosynthesis which results in a necessary increase in energy dissipation by chlorophyll fluorescence.

Basic principles of chlorophyll fluorescence

Any forms of biotic or abiotic stress which have an effect on the photosynthetic capacity of the tree will therefore alter the intensity of the chlorophyll fluorescence emission. Consequently, we can use a Chlorophyll Fluorimeter to measure changes in the extent of fluorescence emission to infer information about the efficiency of light use for photosynthesis.

In essence, the chlorophyll fluorimeter is superbly positioned to quickly and effectively screen for tree deterioration due to a stress related reduction in photosynthetic capacity. What’s more, the technique is fast, non-invasive, non-destructive and can be repeated regularly.

The techniques and methodologies behind the measurement of chlorophyll fluorescence aren’t by any means new.

In 1931, Dr. Hans W. Kautsky (an assistant professor at the Chemisches Institut der Universität in Heidelberg, Germany), noticed that there were characteristic changes in the chlorophyll fluorescence from a leaf when it was subjected to different light intensities. These changes were termed fluorescence induction or the Kautsky Effect.

Dr. Kautsky’s preliminary research on fluorescence induction was performed using the naked eye which was no easy task as chlorophyll fluorescence exists in the far-red spectral band at the upper limit of the eye’s ability to see (photoptic response).

If you were to take a photograph of a tree using an infra-red camera, the tree would appear a purple colour – this is chlorophyll fluorescence. Examples of this can be seen on Google Streetview. The Google cars use filtered infra-red cameras to capture the familiar drivers-eye photos of roads passing through countryside, villages, towns and cities. Occasionally, a filter will break leaving the camera to capture infra-red photos – the image at the top of the screen shows an example of this. Click here to see the Google Streetview page.

The Kautsky Effect

The diagram opposite shows a graph of the intensity of chlorophyll fluorescence over time as originally observed by Dr. Kautsky.

Fortunately, from the late 70’s/early 80’s the electronics industry began to expand rapidly which afforded scientists the luxuries of “hi-tech” instrumentation developed by specialist companies that could measure the Kautsky Effect more efficiently and with far greater detail.

Birth of the PEA

Hansatech Instruments developed the Plant Efficiency Analyser (PEA) Mark 1 in 1989. ThePEA Mk1 was ground breaking in that no other instrument available had ever been able to measure the Kautsky Effect with such high resolution. Measurements from the PEA revealed detailed information about the Kautsky Effect which was previously undetected. This lead to a greater understanding of photosynthesis and how various factors could inhibit different parts of the photosynthetic mechanisms.

PEA Mk2 in use In Monserrat studying he effects of volcanic activity on native tree species

The PEA Mk1 and subsequent PEA Mk2 were enormously successful being employed in photosynthesis research projects the world over. Further advances in technology ensued and before long, the PEA Mk2 was launched followed later by the Handy PEA (2000), Pocket PEA (2007) and more recently, the M-PEA. All the generations of PEA instrument employ the same principles of measurement.

The instruments work by illuminating a dark adapted leaf (zero photosynthetic rate) with an enormously bright red LED typically for 1 second and recording the chlorophyll fluorescence signal that is emitted during this illumination. When the LED is switched on, the leaf instantly (time 0) begins to fluoresce as the photosynthetic apparatus of the leaf begins receiving light energy.

This fluorescence intensity rises rapidly to a maximum level which, in a healthy tree, occurs at approximately 300 – 500 milliseconds. At this point, the photosynthetic apparatus in the leaf is at its maximum photochemical capacity.

Readings at the minimum and maximum chlorophyll fluorescence levels allow us to calculate the maximum Photosynthetic Efficiency of the tree according to its current state of health. Further values taken at other stages during the measurement can also be used to calculate the Vitality of the tree.

Fluorescence and Arboriculture

So how does all this help the Arborist in the field? Chlorophyll fluorescence has the ability to very quickly and easily determine the photosynthetic capacity of a tree according to its current state of health. A reduction in photosynthetic capacity is the first stage in tree deterioration. There are many possible contributing factors to a reduced photosynthetic rate and almost all of them will have put the tree into decline long before we have visual symptoms in the form of leaf yellowing/necrosis or crown/branch dieback.

As previously mentioned, chlorophyll fluorescence measurements are used predominantly in plant physiology research programs which, by definition, all have one key factor – a control sample. Good experimental design and practice dictates that for a comparison to be made, a control sample in optimum condition must first be subjected to the same experimental routine. Once the values are obtained for the control sample, these can be used as a benchmark for the experiment.

Inter-species differences for Fv/Fm

For the Arborist measuring a tree in an urban planting scheme, how do you know what the control values should be? The conventional fluorimeter calculates standard fluorescence parameters which can be used against known constants which give a general “rule-of-thumb” benchmark to which we can relate new results but as the diagram opposite shows, benchmark values can differ between species. The Arborcheck system differs from conventional chlorophyll fluorimeters to allow users to overcome this problem.

Since 2009, Barcham Trees and a team from Bartlett Tree Research Laboratories have committed to annual random testing of over 15,000 individual trees spanning over 275 different species for chlorophyll fluorescence in addition to chlorophyll content and electrolyte leakage to produce a Database of Benchmark Values (DBV) for individual species and cultivar. The DBV has been built in to the Arborcheck system effectively providing the Arborist with the control sample values according to the species being measured.