Productional Biology: Kinetic Imaging of Plant Chlorophyll Fluorescence

Ladislav Nedbalmodified by M. Bartak

Early Fluorescence Imaging 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:

Bio-Sphere2, Tuscon AZ, Nov.29, 2001

Chlorophyll a fluorescence competes with photosynthesis for excitation energy

S0

S1

S2

Chla

hblue

photosynthesis

Fluorescence hNIR

Heat

Role #1 of light in plant fluorescence experiments – measuring light

S0

S1

S2

Chla

photosynthesis

Fluorescence hNIR

Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color.

Achieved by MEASURING light:Typically 10-30s long flashes repeated with a low frequency that

= max

F0=Fmin

Role #2 of light in plant fluorescence experiments – actinic light

S0

S1

S2

Chla

photosynthesis

Fluorescence hNIR

Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color.

Achieved by MEASURING light:Typically 10-30s long flashes repeated with a low frequency that

= (t)

F =F(t)

Role #3 of light in plant fluorescence experiments – saturating light

S0

S1

S2

Chla

photosynthesis

Fluorescence hNIR

Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color.

Achieved by MEASURING light:Typically 10-30s long flashes repeated with a low frequency that

= F =Fmax

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

QA- QA

- QA- QA

- QA- QA

-

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.

QA- QA

- QA- QA

- QA- QA

-

Actinic light is causing fluorescence induction

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

Induction 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

QA-

QA-

QA-QA

-QA

- QA-

QA-

QA-

QA-

QA-

QA-QA

-QA

- QA-

QA-

QA-

PQ-reducing super pulse

Bio-Sphere2, Tuscon AZ, Nov.29, 2001

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

QA-

QA-

QA-QA

-QA

- QA-

QA-

QA-

Fluorescence in PQ-reducing saturation 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

„Cyanobacterial“ Chl fluorescence kinetics

• Source: http://www.sciencedirect.com/science/article/pii/S0014579304014991

Campbell D et al. Microbiol. Mol. Biol. Rev. 1998;62:667-683

Fluorescence emission trace for cyanobacterial quenching analysis.

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’

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

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

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

Bio-Sphere2, Tuscon AZ, Nov.29, 2001