stays almost proportional to the fluorescence quantum yield in most of experimental conditions in which the
fluorescence quantum yield is affected (Fig. 2).
Therefore, from lifetime measurements, a direct estimation of the quantum yield could be obtained through the
relation:
O = x / T 0
where is the quantum yield, z is the mean lifetime and x 0 is the lifetime of fluorescence in the absence of any
other deactivation process. Laboratory chlorophyll fluorescence lifetime measurements on healthy leaves
showed that z ranges from 0.3 to 0.5 ns under moderate daylight conditions, when fluorescence has reached a
stationary level (Fs). However, during a saturating light pulse (Fmax) z increases up to 2 ns [12-13]. Several
methods are known to measure fluorescence lifetimes, among them phase fluorometry, time-correlated single
photon counting (TCSPC) and direct decay measurements after a picosecond excitation are currently in use.
Fluorescence measurements under daylight conditions requires to maximize the i^tio of the fluorescence
emission to the ambient light. Ibis can be done by using a pulsed laser source in ihe sub-nanosecond time
domain [20]. As x is determined from the fluorescence decay within a few nanoseconds, this method is fast and
well adapted to outside measurements under daylight conditions. Thanks to recent improvements on laser
sources and detectors, lifetime measurements in the sub-nanosecond time range are now routine investigation.
In addition, the lifetime parameter has also the advantage to be hardly affected by re-absorption. This
phenomenon is due to the overlap between the chlorophyll fluorescence emission and absorption spectra for
wavelengths < 690 nm. Chlorophyll content of green leaves ranges from 30 to 70 nmole/cm^ of leaf area,
depending on species and age. Ibis amount is synthesized by plants in order to maximize the light absorption.
However, such a concentration is too high for correct spectroscopic measurements. In fact, fluorescence
reabsorption mostly determines the actual shape of the emission spectrum and strongly decreases the apparent
fluorescence yield of green leaves. Interestingly, calculations of the effect of reabsorption on z predict a
negligible lengthening. This has been confirmed by a direct comparison between leaves and a suspension of
diluted isolated chloroplasts [13], [16]. As a consequence, the lifetime parameter appears to be among the most
significant for monitoring the fluorescence properties of intact leaves.
4. DESCRIPTION OF A LIDAR SYSTEM FOR REMOTE MEASUREMENTS OF FLUORESCENCE
DECAYS
Fast Red filter or
photomultiplier interferential 355 nm
i°
Fresnel lens
4 m diameter
coaxial line
\
\L
r -
?
1
M
S= J
I ^'shutter
To
computer
Fast tranzient
analyser 1GHz
Nd:YAG laser
Electronic control
and command
T
35 ps -10 mJ
at 355 nm
telescope |
I
Fluorescence
laser beam ^
and back scattering
6
FIG. 3. Mobile picoseconde LIDAR system of the LURE laboratory for vegetation monitoring.
Eco-physiological applications of fluorescence lifetime measurements must satisfy specific constraints in order
to be useful for plant status monitoring. The distance from the detector to the target together with the presence of
a daylight background are the principal difficulties to overcome. Laboratory techniques for measuring sub
nanosecond fluorescence lifetimes can be hardly extrapolated to field conditions. Remote fluorescence lifetime
