a deeper look at photosynthesis: fluorescence theory and
TRANSCRIPT
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.
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)
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)
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 )
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