Kinetic Imaging of Plant Chlorophyll Fluorescence.

Kinetic Imaging of Plant Chlorophyll Fluorescence

ExperimentKautsky 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

S0

S1

S2

Chla 1

hblue

fluorescence hNIR

photosynthesis


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 plastoquinone-reducing pulses of strong irradiance.

• Typical output is a 2-dimensional map of F0, FM, FV , FV/FM or of NPQ.


Fluorescence

QA-

750 LED’s are on for 10-200 s

Only few PSII RC’s are excited

Yet, sufficient fluorescence emission is produced to capture an image

Measuring flashes have little actinic effects


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- QA- QA

- QA- QA

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During the actinic light exposure, the continuous excitation keeps some of the PSII RC’s closed

LEDs are on for seconds to minutes

Actinic light is causing fluorescence induction


F0

FPEAK

from F0 with open PSII RC’s

to FPEAK with mostly closed PSII RC’s

In fluorescence, the actinic light elicits in plants the Kautsky

effect of fluorescence induction.

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OPEN RC:

QA is oxidized - is LOW

QA PQ=QB

Fe

Pheo

P680YZ

PQPQ

2H2O 4H+

H+

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.2 0.4 0.6 0.8

TIME, s

Fluo

resc

enc

e, r

el.u

nit

Kautsky effect in a diuron-inhibited leaf


QA-

Pheo

P680YZ

PQH2

2H2O4H+

CLOSED RC:

QA is reduced - is HIGH

PQH2

Fe

DCMU

Before the pulse

During the pulse, PSII RC’s are closed by a transient reduction of the plastoquinone pool.

The shutter of the halogen lamp is open typically for 1s

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-QA

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PQ-reducing super pulse


Fluorescence before the pulse

F0

Open PSII reaction centersThe closure of all PS RC’s is reflected by a transient

from F0 to FM.

Fluorescence at the end of the pulseFM

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-QA

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Fluorescence in PQ-reducing super pulse.


0

50

100

150

200

250

-10 0 10 20 30 40 50 60 70 80

TIME, seconds

FLU

OR

ES

CE

NC

E, r

.u.

F0

FM

FV

FS

FM’

Pixel-to-pixel arithmetic image operations

Color photograph

Fluorescence FM image

Chlorophyll fluorescence from ripe lemon fruits

Color photograph

Fluorescence images

F0 FV FM FV/FM

Heterogeneous lemon pigmentation

Color photograph

Fluorescence images

F0 FM FV/FMFV

Post-harvest lemon damage

Green mold infection of lemons

Color photographs

Flu

ores

cenc

e

Time after infection 0 h 48 h66 h

84 h

0

0.005

0.01

0.015

0.02

0.025

0.03

0 50 100 150 200

Fluorescence intensity, r.u.

Re

lativ

e fr

eq

uenc

y, r

.u.

Histograms reveal parameters of the highest contrast

FM

F0

FV

The mold-infected peel tissue contains detached antenna complexes

0

0.2

0.4

0.6

0.8

1

640 660 680 700 720 740 760 780

Wavelength, nm

Flu

ore

sce

nce

, r.u

. healthy

infected

Phytotoxin response visualized by fluorescence

Sinapis alba60 h, 2000 mg/l destruxin

Brassica oleracea60 h, 0-500 mg/l destruxin

0.05 mg/l

0 mg/l

0.5mg/l50mg/l

500mg/l

TIME, s0 5 20 30 40

PLA

NT

FLU

OR

ES

CE

NC

E, F

(t) / F

o

0

2

4

6

8

10Hcf mutant

WT

Saturating Pulses:

ONActinic Light: OFF

A B

Mutant selection

High-light stress sensitivity

FV/FM

2


TIME, s0 5 20 30 40

LE

AF

FL

UO

RE

SC

EN

CE

, F(t

) / F

o

0

1

2

3

4

5

6

7

Saturating Pulses:

ON

Young Leaf

Old Leaf

A B C

Actinic Light:

OFF

Leaf development

T I M E , s

0 5 1 0 1 5 2 0 2 5

SIG

NA

L, r

el.u

nits

5 0

5 5

6 0

6 5

7 0

I R R A D I A N C E , m o l ( p h o t o n ) . m - 2 . s - 1

0 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0

FL

UO

RE

SC

EN

CE

F(t

), F

M',

rel.u

nits

2

4

6

8

1 0

1 2

1 4

1 6

1 8

A

C

BA

O NA c t i n i c L i g h t : O F F

Field operation

0 100 110 120 130 140 1500

20

40

60

80

100 Elodea cell diatom

rela

tive

fluor

esce

nce

time / s

50 m

FV / FM FS

FS – F0

Microscopic kinetic fluorescence imaging

diatoms

Elodea chloroplasts

Average

Heterocyst formation in cyanobacteria

Transmittance FM fluorescence

Kinetic Imaging of Plant Chlorophyll Fluorescence.

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