Kinetic Imaging
of Plant Chlorophyll Fluorescence
Experiment
Kautsky and Hirsch (1931) irradiated a dark-adapted leaf with a blue light
and observed it visually through a dark-red glass. Here is a high-tech
presentation of what they saw:
© Photon Systems Instruments, spol. s r.o., CZ
Chla fluorescence competes with
photosynthesis for excitation energy
S2
hnblue
S1
photosynthesis
fluorescence hnNIR
Chla 1
S0
© Photon Systems Instruments, spol. s r.o., CZ
FluorCam of Photon Systems Instruments, Ltd.
•
FluorCam was designed to measure heterogeneity of plant photosynthetic activity
among species, mutant plants or within a single plant from the whole plant level
down to individual cells. Localized biotic or abiotic stress is a typical factor
causing heterogeneity in the plant activity that can be visualized by FluorCam.
•
FluorCam measures fluorescence image transients of leaves, small plants, and of
algal or cyanobacterial colonies. Typical size of an investigated object is smaller
than 10  13 cm
•
The measured fluorescence is excited by short measuring flashes.
•
The transients are induced by a continuous actinic light and by plastoquinonereducing pulses of strong irradiance.
•
Typical output is a 2-dimensional map of F0, FM, FV , FV/FM or of NPQ.
© Photon Systems Instruments, spol. s r.o., CZ
Measuring flashes have little actinic effects
750 LED’s are on for 10-200 ms
Only few PSII RC’s are excited
Fluorescence
QA-
Yet, sufficient fluorescence
emission is produced
to capture an image
© Photon Systems Instruments, spol. s r.o., CZ
Actinic light is causing fluorescence induction
LEDs are on
for seconds to minutes
QA- QA- QA-
QA- QA- QA-
During the actinic light exposure, the continuous
excitation keeps some of the PSII RC’s closed
© Photon Systems Instruments, spol. s r.o., CZ
In fluorescence, the actinic light
elicits in plants the Kautsky
effect of fluorescence induction.
FPEAK
QA- QA- QA-
F0
QA- QA- QA-
to FPEAK with mostly closed PSII RC’s
from F0 with open PSII RC’s
© Photon Systems Instruments, spol. s r.o., CZ
Kautsky effect in a diuron-inhibited leaf
OPEN RC:
QA is oxidized - is LOW
CLOSED RC:
QA is reduced - is HIGH
1 .4
QA
Fe
Pheo
P680
YZ
2H2O
PQ=QB
PQ
PQ
Fluor esce nce , re l.un it
1 .2
H+
1
Fe
QA-
0 .8
0 .6
Pheo
0 .4
P680
0 .2
PQH2
PQH2
YZ
0
4H+
DCMU
0
0 .2
0 .4
T I M E, s
0 .6
0. 8
2H2O
4H+
© Photon Systems Instruments, spol. s r.o., CZ
PQ-reducing super pulse
The shutter of the halogen
Before
thetypically
pulse for 1s
lamp
is open
Q A-
QA-
QA-
QA-
QA-
QA-
QA-
QA-
During the pulse, PSII RC’s are closed
by a transient reduction of the plastoquinone pool.
© Photon Systems Instruments, spol. s r.o., CZ
Fluorescence in PQ-reducing super pulse.
FM
Fluorescence at the end of the pulse
The closure of all PS RC’s
is reflected by a transient
from F0 to FM.
Open PSII reaction centers
QA-
QA-
QA-
QA-
QA-
QA-
QA-
QA-
F0
Fluorescence before the pulse
© Photon Systems Instruments, spol. s r.o., CZ
FLUORESCENCE, r.u.
Pixel-to-pixel arithmetic image operations
250
FM
200
150
100
FM’
FV
FS
50
0
F0
-10 0
10 20 30 40 50 60 70 80
TIME, seconds
Chlorophyll fluorescence
from ripe lemon fruits
Color photograph
Fluorescence FM
image
Heterogeneous lemon pigmentation
Color photograph
F0
FV
FM
FV/FM
Fluorescence images
Post-harvest lemon damage
Color photograph
F0
FV
FM
FV/FM
Fluorescence images
Green mold infection of lemons
Fluorescence
Color photographs
Time after infection
0h
48 h
66 h
84 h
Histograms reveal parameters
of the highest contrast
Relative frequency, r.u.
0.03
0.025
0.02
FV
F0
0.015
FM
0.01
0.005
0
0
50
100
150
Fluorescence intensity, r.u.
200
The mold-infected peel tissue contains detached
antenna complexes
Fluorescence, r.u.
1
healthy
0.8
0.6
0.4
0.2
infected
0
640 660 680 700 720 740 760 780
Wavelength, nm
Phytotoxin response visualized by
fluorescence
50mg/l
0.5mg/l
0.05 mg/l
0 mg/l
Sinapis alba
60 h, 2000 mg/l destruxin
500mg/l
Brassica oleracea
60 h, 0-500 mg/l destruxin
Mutant selection
10
B
Hcf mutant
8
6
4
2
WT
0
5
20
TIME, s
30
0
40
Saturating Pulses:
Actinic Light:
ON
OFF
PLANT FLUORESCENCE, F(t) / Fo
A
High-light stress sensitivity
FV/FM
2
© Photon Systems Instruments, spol. s r.o., CZ
Leaf development
7
6
Young
Leaf
5
A
B
C
4
3
Old
Leaf
LEAFUORESCN,F(t)/o
2
1
0
5
2
0
3
0
T
I
M
E
,
s
0
4
0
Saturating Pulses:
OFF
Actinic Light:
ON
Field operation
TIME, s
5
10
15
20
A
25
B
70
65
60
55
50
Actinic Light:
OFF
ON
FLUORESCENCE F(t), FM', rel.units
18
16
14
12
10
8
6
C
4
2
0
250
500
750
1000
1250
-2 -1
IRRADIANCE, mmol(photon).m .s
1500
SIGNAL, rel.units
0
Microscopic kinetic
fluorescence imaging
FV / FM
FS
relative fluorescence
100
Elodea cell
diatom
80
60
40
20
0
0
100
110
120
130
140
150
time / s
FS – F0
50 mm
diatoms
Average
Elodea
chloroplasts
Heterocyst formation in cyanobacteria
Transmittance
FM fluorescence