6.

INTRODUCTION
Chlorophyll looks green in white light because it absorbs light in the blue (round
about 420 nm) and in the red parts (round about 660 nm) of the visible spectrum and
transmits and reflects in the green. Light can be regarded as a stream of particles, or
parcels, of energy. Each particle (quantum or photon) can bring about a single
photochemical event provided that it carries sufficient energy to drive that specific
event. Each quantum of red light which is absorbed by a chlorophyll molecule raises
an electron from a ground state to an excited state and all of its energy is transferred in
this process. This excitation is, essentially, an oxidation. Electron transport is
initiated, as the electron is lifted into a higher energy orbital and a positively charged
“hole” is left behind. Absorption of blue light causes even greater excitation (because
of the higher energy content of the blue quantum) but the elevated electron then falls
back into the “red orbital too quickly to permit useful chemical work. Thus, whatever
the quality of the light absorbed, the electron reaches the same energy level more or
less immediately after excitation and all subsequent events derive from this common
starting point (“excited state one”).
Figure 6.1. The excitation of chlorophyll by light.
The parallel lines represent energy sub-states or electronic orbitals. Thus the energy
delivered by the absorption of a blue photon (left) is sufficient to raise on electron to
“excited state two” from where it rapidly returns by a process of radiationless
de-excitation, “cascading” through sub-states, to excited state “one”. A photon of
red light (centre) only has enough energy to raise an electron to excited state “one”
but this excited state is sufficiently stable to permit useful chemical work and is, in
effect, the starting point of all other events in photosynthesis. “Excited state one” con
also dissipate energy by re-emitting light as (deep red) fluorescence.
blue
hv
red
hv
hv fluorescence
}Ground state
} Excited state (one)
} Excited state (two)
Radiationless
de-excitation
Chemistry
Chlorophylls a and b
H2C = CH
H3C
H3C
H
CH2
CH2
C=O
O
Phytyl

radiationless de-excitation and some is used to drive the “chemical” reactions of

photosynthesis (ATP synthesis, NADP reduction etc). Some energy is also dissipated

as fluorescence. This, it should be emphasised, is not reflected or transmitted light. It

is light which is created in the leaf, just as electron transport (electric current) through

the filament of an incandescent lamp leads to the emission of light. Fluorescence

(which takes about 10-9 sec to discharge) derives from the lower more persistent

“excited state one” rather than “excited state two” which decays in about 10-13 sec.

For this reason chlorophyll fluorescence is red, regardless of the quality of the

exciting light and it is a deeper (longer wavelength) red than the red absorption peak

because of the “Stokes shift” (the rapid cascade of heat dissipation which occurs

within “excited state one”) so that electrons fall to the ground state from the lowest

levels of excitation and must therefore give rise to photons of lower energy content

(i.e. longer wavelength light).

7. PRINCIPLE OF MEASUREMENT

The fraction of excitation energy which is dissipated as fluorescence in vivo is very

small (3-5%). In solution this fraction is much larger (up to 30%) and if a solution of

chlorophylls in ethanol, or acetone, is illuminated in a rectangular vessel it will look

green from the front and deep red from the side. (Chlorophyll reflects in the red and

the blue and transmits in the green but, viewed from the side, the retina is no longer

flooded with green photons and the deep red fluorescence emanating from the

excited chlorophyll is readily perceived). Photomultipliers are often used in light

measurements but, in most circumstances, fluorescence from a leaf can be adequately

detected with a photodiode. As in photosynthesis, each photon which falls on the

detector initiates one photochemical event in which an electron is raised to a higher

energy level and a positively charged “hole” is created. In photosynthesis, the “hole”

accepts an electron from water (Figs. 8.1 and 8.2) and the electron is passed (via

photosystem II and I) to NADP. In the photodiode, the corresponding electron

transport (electric current) is amplified and can be applied, as a voltage, to a

pen-recorder. The main problem is to prevent the detector “seeing” light which is not

fluorescence and, inevitably, a relatively large fraction of the “actinic” light (the light

used to drive photosynthesis) will be reflected from the leaf surface into the detector.

Accordingly, the detector is protected by optical filters which, ideally, exclude all of

the reflected actinic light and transmit all of the fluorescence. In practice a

compromise is needed because in terms of “energy-effectiveness” (Section 6) red

light is best for photosynthesis and the peak of chlorophyll a absorption in the red

(about 680 nm) is not far removed from the peak of chlorophyll a fluorescence (about

685 nm). For this reason blue exciting (actinic) light is sometimes used because it is

readily separated from the fluorescence peak. For many purposes, however,

fluorescence signals at longer wavelengths (i.e. wavelengths about 740 nm, where

there is a smaller fluorescence maximum) are sufficiently strong (and sufficiently

similar in quality to those at shorter wavelengths) to allow the employment of filters

which will exclude most of the actinic light and transmit most of the fluorescence. In

many experiments, fluorescence

Pale

Dark

690 740

Fluorescence spectra of

pale and dark leavesafter

French and Young,

1952