R.L. Garcia October 2015

LI-COR BioSciences

A Deeper Look at Photosynthesis:

Fluorescence Theory and Practice

Using The LI-6400XT

Copyright © 2014 LI-COR Biosciences, Inc. All rights reserved.

How do we measure photochemistry?

Gas exchange: CO2 and H2O

Chlorophyll Fluorescence

Chlorophyll Fluorescence

F + H + P = 1

The absorption spectrum of chlorophyll

4

If we assume the ratio of heat to fluorescence de-excitation does not change,

H/F = Hm/Fm (eq. 5)

H = F(1-Fm)/Fm (eq. 6)

We can solve for H & P if we measure F in non-saturating light (F) and saturating

light (Fm)

P = 1-F-H (eq. 7)

P =1 - F - [F(1-Fm)/Fm] (eq. 8)

P = Fm-F/ Fm (eq. 9)

F + H + P = 1 (eq. 1) At saturating light intensity:

No increase in P with further increase in light intensity and F & H maximum

F = Fm, H = Hm, P = 0 (eq. 2)

Fm + Hm + 0 = 1 (eq. 3)

Hm = 1 – Fm (eq.4)

P = Fm-F/ Fm

Murchie and Lawson 2013

modulated LED

650 nm short

pass filter

pulsed-fluorescence

(~630-850 nm)

RG-9

700 nm long

pass filter

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

200 300 400 500 600 700 800 900 1000 1100

electronics

detector scattered light

Pulsed amplitude modulation fluorescence

low intensity

modulated

light

F modulated

fluorescence

F(minimum

fluorescence yield)

PSII

2 H2O O2+4e-+4H+

high intensity

light flash

Fm’

Fm ’ (maximum

fluorescence yield)

LCF Design

• Red (630nm), Blue

(470nm), Far red

(740nm) LED’s

• Fluorescence

Detection at 715nm

Fm’

NPQ

ETR qE

ETR vs. AG

gm

qE vs. ETR

PSII

=

(Fm’-F)

Fm’

= (PSII*i**fII)

A

Ci – Γ* [ETR + 8 (A + Rd)]

ETR – 4 (A + Rd)

=

(Fm-Fm’)

Fm’ =

(Fm”-Fm’) Fs

Fm’ Fm’ = *

Cc

AN Vcmax climate

modeling Cc

Fm’

NPQ

ETR qE

ETR vs. AG

gm

qE vs. ETR

PSII

=

(Fm’-F)

Fm’

= (PSII*i**fII)

A

Ci – Γ* [ETR + 8 (A + Rd)]

ETR – 4 (A + Rd)

=

(Fm-Fm’)

Fm’ =

(Fm”-Fm’) Fs

Fm’ Fm’ = *

Cc

AN Vcmax climate

modeling Cc

Underestimation of Fm’ can be inferred

0.5 to 1 second

m

ol m

-2 s

-1

6,200

7,600

4,800

3,400

8,500

hypothetical saturation of AFm’

approaching

saturation

Extrapolated Fm’ (EFm’) or true Fm’

0

11% 8% 6% 5% 4%

Apparent Fm’ or AFm’

AFm’

infinite irradiance

Multiphase FlashTM fluorescence

Used to measure Fm’ at infinite irradiance

AFm’

Flu

ore

sce

nce

yie

ld (

F)

Irra

dia

nce

(µ

E)

Phase 1 Phase 2 Phase 3

Irra

dia

nce

(µ

mo

l m

-2s

-1)

AFm’

F (

Ph

ase

2)

1/Phase 2irradiance (m2 s mol-1) *104

10%

Extrapolated Fm’ ~ true Fm’

infinite irradiance

60

% R

am

p

500 ms

Ramp rate = mol photons m-2 s-2

Loriaux et, al., 2013

Rectangular Flash Method

y0 = 38.7 + 7.7 b = 2.9 + 0.2

MultiPhase Flash Method

y0 = 10.2 + 8.9 b = 4.7 + 0.2

Underestimates of Fm’ result in

underestimates of electron transport

Flexas, J. et al. Plant, Cell, and Environment 2008

‘J’ corresponds to an estimate of electron transfer obtained by chlorophyll fluorescence

Exploring deeper using gas exchange and fluorescence

gm =A

Ci -G*(J +8(A+Rd ))

J - 4(A+Rd )

Fluorometer Display Changes

Fluorometer Function Key Changes

Fluorescence Instrument Basics

• Higher fluorescence emission, better signal: noise

• Higher excitation intensities, higher fluorescence emission

• Higher excitation frequencies, higher excitation intensity

• Calculated parameters like Fv/Fm are not highly influenced by fluorescence emission intensities (they are unitless)

• To compare across time, emission intensities do matter

Fluorometer Settings

Fluorometer Settings

Fluorometer Settings

Fv/Fm

DF/Fm’

Driving thru Zambia

Photo by R.L. Garcia, 1984

Thank you