Table 2. Comparison of lifetimes (x) and fractional intensities (/) of the different kinetic components at
decreasing levels of complexity of the leaf derived samples. For simplicity of presentation standard
deviations were omitted from the table. They were always less than 10% of the mean. The number of
samples is indicated in parentheses. The excitation wavelength was 360 nm.
sample
type (n)
emission
kinetic component
average
lifetime
wavelength
fast
medium
slow
very slow
x (ns)
/(%)
x (ns)
/(%)
x (ns)
/(%)
x (ns)
/(%)
xm (ns)
leaf adaxial (4)
460
0.3
37
1.1
36
3.2
19
7.6
8
1.7
leaf abaxial (3)
460
0.3
36
1.0
30
3.0
24
7.9
10
1.9
mesophyll (11)
460
0.3
32
1.0
31
3.3
20
8.8
17
2.6
chloroplasts (4)
460
0.4
27
1.6
39
4.5
27
11
7
2.7
leaf (adaxial) (4)
520
0.3
27
1.2
31
3.4
29
8.5
13
2.5
leaf (abaxial) (3)
520
0.3
28
1.1
32
3.4
30
8.7
10
2.3
mesophyll (12)
520
0.3
33
1.0
31
3.3
23
9.2
13
2.4
chloroplasts (5)
520
0.4
26
1.6
38
4.4
29
13
7
2.8
Table 3. Effect of wavelength combination on the lifetimes and fractional intensities of the four kinetic
components in sugar beet mesophyll.
wavelength kinetic component
excitation
emission
fast
medium
slow
very slow
lifetime
t(ns)
/(%)
t(ns)
/(%)
t(ns)
/(%)
t(ns)
/(%)
xm (ns)
340
430
0.29
30
1.1
29
3.8
18
8.3
22
3.0
375
525
0.32
32
1.3
32
4.2
24
11.2
12
2.6
420
540
0.12
24
1.4
35
4.4
32
9.8
8
2.7
3.2.Comparison of the contribution of different kinetic components to the fluorescence at
a fixed wavelength.
An alternative approach to DAS is to compare the characteristics of the kinetic components of
fluorescence at fixed wavelength combinations. This permits the comparison of lifetimes and contributions of the
four kinetic components to fluorescence in samples of decreasing complexity. Four decay components can be
resolved in all types of samples and at any given wavelength examined. It is reasonable to assume that all four
fluorescence decay components are complex sums of kinetic contribution from different fluorophores having
different lifetimes, if we keep in mind the complexity of a leaf or even chloroplasts. Still, each of the decay
components represents a class of fluorophores having similar lifetimes: short (0.3-0.4 ns), medium (1-1.5 ns ),
long (3-4,5 ns ) and very long (8-11 ns). The four resolved decay components can be treated accordingly for
comparative purposes by analyzing the variation of the respective fractional contribution in different types of
sample, and by comparing it with the spectral characterization described above. The lifetimes and fractional
contributions of the four kinetic components to the total fluorescence in different types of sample are presented in
Table 2. The excitation wavelength was fixed at 360 nm and the emission centered at 460 or 520 nm, so the
variations of the blue and green emitting fluorophores in different types of sample could be compared, including
isolated chloroplasts. Chloroplasts were more fragile and their fluorescence much weaker than that of leaves and
mesophyll therefore DAS could not be properly recorded with the actual set up.
In Table 3, it can be seen that the lifetimes of the four components are very similar in leaves and
mesophyll, both in the blue and green, but they were all longer in chloroplasts. This permits direct comparisons
of fractional intensities in leaves and mesophyll, but one should be more cautious when comparing them to
chloroplasts. Still, in all samples the slow component had a significantly larger fractional contribution in the
green (520 nm) than in the blue (460 nm) without changes in lifetime which is close to that of flavins. The
lifetime and fractional contribution of the medium component did not show any significant changes between
leaves and mesophyll, in blue and green, allowing no other conclusions apart that this component is the
dominant one in chloroplasts. Along with the decrease in complexity, from leaves to chloroplasts, there was a
