National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
Using chlorophyll fluorescence to study photosynthesis
葉綠素螢光—光合作用之探討
1. Introduction
1.1 Chlorophyll fluorescence
In the photosynthetic apparatus, light is absorbed by the antenna pigments such as chlorophyll,
and the excitation energy is transferred to the reaction centers of the two photosystems (Fig.1).
Then the energy drives the primary photochemical reactions that initiate the photosynthetic energy
conversion. Under optimal conditions, more than 90% of absorbed light quanta are utilized by
photosynthesis. A minor competing process of deactivation of excited pigments is the emission of
chlorophyll fluorescence of photosystem II (PSII) (Fig. 2).
Thylakoid membrane electron transport chain
ATP
ADP
NADPH
h
h
NADP+ + H+
2 H+
CF1
Fdx
n H+
Fdx
Cytb6f
PSII
PQ
PSI
e-
PQH2
e-
CF0
e-
e-
PC
PC
2 H+
H2 O
1
2
O2 + 2 H+
Buchanan et al., 2000
Fig. 1 Thylakoid membrane electron transport chain.
Schematic view of energy conversion and
electron transport in photosynthesis
hv
hv
LHC I
LHC II
Heat
Fluorescence
H2O
Z
PS I
PS II
ChlaII*
P680+
Phe-
Heat
ChlaI*
Fluorescence
QA
QB
PQ
Cytb/f
PC
P700
Pd
NADPH
Calvin
cycle
Heat
Heat
Fig. 2 Energy conversion in photosynthesis
Chlorophyll Fluorescence/ Page-1
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
Though the fraction of excitation energy which is dissipated as fluorescence in vivo is very
small (3-5%), the changes of the electron transport rate can be quantified by the fluorescence
emission. Generally, fluorescence yield is high when photochemistry and heat dissipation are lowest.
Therefore, changes in the fluorescence yield reflect changes in photochemical efficiency and heat
dissipation. Chlorophyll fluorescence allows us to study the different functional levels of
photosynthesis indirectly (e.g. processes at the pigment level, primary light reactions, thylakoid
electron transport reactions, and etc.).
1.2 Chlorophyll fluorescence spectra and parameters
A typical measurement on an intact leaf by saturation pulse method is shown in Figure 3. The plant
was dark adapted for 20 min prior to the measurement. Upon the application of a saturating flash,
fluorescence raises for the ground state value, Fo, to its maximum value, Fm. Therefore, the
maximum quantum efficiency of PSII primary photochemistry can be determined as Fv/Fm
(Fv=Fm-Fo), representing the photosynthetic activity. A change of Fo can be interpreted as a
change of the rate constant of energy trapping by PSII center that could be the result of a physical
distortion of LHCII. If the plant was light adapted, a quenching of fluorescence occurs and the
fluorescence yield reaches a steady state value (Fs). Upon the application of a second saturation
flash in the presence of actinic light, the maximum fluorescence obtained (Fm’) is lower to that
observed in the dark (Fm). A light-adapted quantum efficiency of photosystem II (PSII) can be
obtained as (Fm’-Fs) / Fm’.
Chlorophyll Fluorescence Spectra
Dark-adapted
Light-adapted
Fm
Fm’
Fv
Fv’
Fs
Fo
Saturating pulse
Saturating pulse
0
Measuring beam
Measuring beam
Popp, 2000
Figure 3. Chlorophyll fluorescence spectra
There are some useful chlorophyll fluorescence parameters listed as follows:
Fo:
dark-adapted minimal chlorophyll fluorescence
Fm:
dark-adapted maximal chlorophyll fluorescence
Fv:
Fv/Fm:
dark-adapted variable chlorophyll fluorescence (Fv = FmFo)
dark-adapted maximum quantum efficiency of photosystem II [Fv/Fm = (Fm-Fo) / Fm]
Chlorophyll Fluorescence/ Page-2
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
Fs:
light-adapted steady state fluorescence yield
Fm':
light-adapted maximal chlorophyll fluorescence
Fv':
PSII:
light-adapted variable chlorophyll fluorescence (Fv' = Fm'Fo')
light-adapted quantum efficiency of photosystem II [ PSII = (Fm’-Fs) / Fm’]
2. Experiments
2.1 Determination of chlorophyll content
2.1.1 Principle:
(1) Chlorophyll a mainly absorbs at wavelength 663nm and chlorophyll b mainly absorbs at
wavelength 645nm; however, there is an overlap absorbance which need to be subtracted.
(2) Calculation based on Mackinney-Arnon equations with dilution factors.
Total Chl = Chl a + Chl b = (4.0 x A665nm) + (25.5 x A650nm) (g Chl/mL)
2.1.2 Equipment
(1) Centrifuge (Sigma 202 MK)
(2) UV-VIS spectrophotometer
2.1.3 Supplies
(1) Centrifuge tubes, glass tubes, cuvette
(2) Pasteur pipettes, pipettemen, tips
2.1.4 Materials
Green alga Chlorella sp. DT or Chlorella sp. 8b in Chlorella medium were routinely cultured
in column at 32C±2C with continuous light illumination of 120 E m-1 s-1 and bubbling of
4% CO2.
2.1.5 Solutions
(1) Methanol
(2) Arnon's medium
2.1.6 Procedures:
(1) Extract chlorophyll from green alga Chlorella.
(a) Collect 5 mL Chlorella culture in a plastic centrifuge tube in duplicates.
(b) Centrifuge at 3,000 rpm for 3~5 min in a Sigma table centrifuge. Discard supernatant.
(c) Add 5 mL Methanol and whirllingmix for few minutes.
(d) Then incubate at 60C for 5 min.
(e) Centrifuge at 5,000 rpm (Sigma 202 MK) for 5 min. Remove supernatant into a new
10-mL glass tube.
(2) Measure A665nm and A650nm (A: Absorbance) using UV-VIS spectrophotometer.
Blank: Pour 2 mL of methanol into glass cuvette as ‘Blank’.
Sample: Pour 2 mL of methanol-extract into glass cuvette.
Chlorophyll Fluorescence/ Page-3
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
(3) Reading of A665nm and A650nm
Measurement
A665nm
A650nm
Blank
m=
n=
Sample-1
r1 =
q1 =
Sample-2
r2 =
q2 =
Average
r = (r1 + r2)/2=
q = (q1 + q2)/2=
(SampleBlank)
S=mr
T=nq
(4) Calculation:
If
Then
A665nm = S
A650nm = T
Total Chl = Chl a + Chl b
= (4.0 x S) + (25.5 x T) (g Chl /mL)
= W (g Chl /mL)
(5) Make-up 4 tubes containing 5 mL suspension of 4 and 8 g Chl /mL each.
Sample-A: 4 g Chl /mL  20 mL = (W g Chl /mL  ____ mL) + Arnon's  ____ mL
Sample-B: 8 g Chl /mL  20 mL = (W g Chl /mL  ____ mL) + Arnon's  ____ mL
2.2 Measurement of chlorophyll fluorescence parameters
2.2.1Equipment
(1) Hasantech Chlorophyll Fluorometer
(2) Projector
(3) Incubator
2.2.2 Supplies
(1) Cuvette, glass tubes
(4) Ice box
2.2.3 Procedures
(1) Samples treatment
(a) Short-term:
(i)
Cold treatment at 0C for 30 min: Put the glass tubes of Chlorella into ice box
(ii)
Light treatment with high light (>1000 E m-1 s-1) for 30 min: Turn on projector
(b) Long-term:
(i)
Cold treatment with 15C for a couple of days: Chlorella were cultured in
column with continuous light illumination and sufficient CO2.
(2) Dark-adapted or Light-adapted treatment
(a) Light-adapted treatment: Samples are led on the bench at room temperature.
(b) Dark-adapted treatment: Samples are dark-adapted for a period of 10 min at room
temperature.
(3) Measure Fo, Fv/Fm, … parameters in duplicates and record in the data sheet.
Chlorophyll Fluorescence/ Page-4
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
3. Results
3.1 Data sheet:
(1) Short-term treatment
1-A. Control: 4 g Chl /mL
Sample Fo
Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Fv/Fm
1-B. Control: 8 g Chl /mL
Sample Fo
Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Fv/Fm
2-A.Cold treatment / 0C for 30 min treatment: 4 g Chl /mL
Sample Fo
Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Fv/Fm
2-B. Cold treatment / 0C for 30 min treatment: 8 g Chl /mL
Sample Fo
Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Fv/Fm
3-A. Light treatment / 1000 E m-1 s-1 for 30 min: 4 g Chl /mL
Sample Fo
Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Fv/Fm
3-B. Light treatment / 1000 E m-1 s-1 for 30 min: 8 g Chl /mL
Sample Fo
Fm
Fv/Fm
Fm’
PSII
Fv/Fm
Sample Fo
Sample Fs
Fm
Sample Fs
Fm’
PSII
Chlorophyll Fluorescence/ Page-5
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
(2) Long term treatment at 15C: Chlorella DT vs 8b
Day-0
Culture-A: 4 g Chl /mL
Sample
Fo
Culture-B: 8 g Chl /mL
Fm
Sample
Fv/Fm
Sample
Fo
Fo
Fm
Fv/Fm
Fm
Sample
Fv/Fm
Fo
Fm
Fv/Fm
Day-2
Culture-A:
Sample
Fo
Culture-B:
Fm
Sample
Fv/Fm
Sample
Fo
Fo
Fm
Fv/Fm
Fm
Sample
Fv/Fm
Fo
Fm
Fv/Fm
Day-4
Culture-A:
Sample
Fo
Culture-B:
Fm
Sample
Fv/Fm
Sample
Fo
Fv/Fm
Fo
Fm
Fv/Fm
Fm
Sample
Fo
Fm
Fv/Fm
Continuously record the data in the following every two days if required.
Then plot the relationships of [Chl] versus Day and Fv/Fm versus Days from long-term treatment
experiment?
3.2 Questions:
(1) How do the chlorophyll parameters change under the stresses of low temperature and high
light?
(2) What information is obtained from the treatment of low temperature and high light?
Chlorophyll Fluorescence/ Page-6
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
4. Answers
4.1 Chlorella suspension
(1) Readings of A665nm and A650nm:
Measurement
A665nm
A650nm
Blank
m = 0.000
n = 0.009
Sample-1
r1 = 0.483
q1 = 0.265
Sample-2
r2 = 0.484
q2 =0.264
Average
r = (r1 + r2)/2 = 0.484
q = (q1 + q2)/2 = 0.256
(SampleBlank)
S = m  r = 0.484
T = n  q = 0.256
(2) Calculation:
If
A665nm = 0.484
A650nm = 0.256
Total Chl = (4.0  A665nm) + (25.5  A650nm) (g Chl/mL)
= (4.0  0.484)
+ (25.5  0.256) (g Chl/mL)
= 8.464 (g Chl/mL)
Final Total Chl = 4 (dilution factor)  8.464 (g Chl/mL) = 33.86 (g Chl/mL)
Then
(3) Make-up 4 tubes containing 5 mL suspension of 4 and 8 g Chl /mL each.
Sample: 4 g Chl /mL  20 mL = (33.86 g/mL 
Sample: 8 g Chl /mL  20 mL = (33.86 g/mL 
4.2 Chlorophyll Parameters
4.2.1 Short-term treatment
1-A. Control: 4 g Chl /mL
Sample Fo
70
Fm
Fv/Fm 0.840
Sample Fo
0
Fm
Fv/Fm 0.840
1-B. Control: 8 g Chl /mL
Sample Fo
181
Fv/Fm 0.813
Sample Fo
142
Fv/Fm 0.815
Fm
Fm
2.4
4.8
438
Sample Fs
438
PSII
Sample Fs
PSII
969
Sample Fs
769
PSII
Sample Fs
PSII
2-A.Cold treatment / 0C for 30 min treatment: 4 g Chl /mL
Sample Fo
73
Fm 411
Sample Fs
Fv/Fm 0.822
PSII
Sample Fo
69
Fm 412
Sample Fs
Fv/Fm 0.832
PSII
2-B. Cold treatment / 0C for 30 min treatment: 8 g Chl /mL
Sample Fo
181
Fm 969
Sample Fs
Fv/Fm 0.813
PSII
Sample Fo
142
Fm 769
Sample Fs
Fv/Fm 0.815
PSII
mL) + Arnon's 
mL) + Arnon's 
17.6
15.2
90
0.811
80
0.818
Fm’
478
Fm’
441
184
0.798
170
0.797
Fm’
184
Fm’
170
90
0.811
80
0.818
Fm’
478
Fm’
441
184
0.798
170
0.797
Fm’
478
Fm’
441
mL
mL
Chlorophyll Fluorescence/ Page-7
National Chung-Hsing University
Biophysics Practical 2003 / L-F Chien
3-A. Light treatment / 1000 E m-1 s-1 for 30 min: 4 g Chl /mL
Sample Fo
90
Fm 311
Sample Fs
Fv/Fm 0.710
PSII
Sample Fo
98
Fm 176
Sample Fs
Fv/Fm 0.443
PSII
-1 -1
3-B. Light treatment / 1000 E m s for 30 min: 8 g Chl /mL
Sample Fo
207
Fm 319
Sample Fs
Fv/Fm 0.351
PSII
Sample Fo
189
Fm 342
Sample Fs
Fv/Fm 0.447
PSII
121
0.620
107
0.405
Fm’
319
Fm’
180
239
0.282
231
0.345
Fm’
333
Fm’
352
4.2.2 Long-term treatment
0.9
0.9
0.8
0.8
0.7
0.7
Fv/Fm
Fv/Fm
32oC
0.6
0.5
0.6
0.5
8b(4 g/ml)
8b(8 g/ml)
DT(4 g/ml)
DT(8 g/ml)
0.4
8b(4 g/ml)
8b(8 g/ml)
DT(4 g/ml)
DT(8 g/ml)
15oC
0.4
0.3
0.3
0
1
2
3
Days
4
5
6
0
2
4
6
8
10
12
14
16
Days
4.3 Answers to the questions
(1) How do the chlorophyll parameters change under the stresses of low temperature and high
light?
Answer:
(a) The counts of Fo and Fm were proportional to the concentration of Chlorella.
(b) The ratio of Fv/Fm and PSII were slightly decreased from 0.84 to 0.81 when the
Chlorella cells were treated with low temperature of 0C for 30 min.
(c) The ratio of Fv/Fm was decreased from 0.84 of the control to 0.35 of the
high-light-treated Chlorella. The PSII was decreased from 0.62 of the control to
0.28 of the high-light-treated Chlorella.
(2) What information is obtained from the treatment of low temperature and high light?
Answer:
(a) Photosynthetic efficiency was significantly reduced by the treatment of high light
in short term (30 min), but not by that of low temperature.
(b) During long-term of cold treatment at 15C, Fv/Fm was decreased to 0.48 at fourth
day, compared with the control of 0.84 at 32C.
Chlorophyll Fluorescence/ Page-8