ETR Measurement with FluorCam
Introduction
ETR, or electron transport rate, is a light-adapted parameter that is directly related to PSII operating
efficiency (Y(II), Genty parametr, Fq´/Fv´, φPSII) by the equation, ETR = φPSII × PAR × aL × f PSII, where:
φPSII= operating quantum efficiency of PSII
PAR = photosynthetically active radiation (in µmol/m2/s)
aL= leaf light absorption (between 0 and 1, usally about 0.8)
f PSII= proportion of PSII over[PSII + PSI] (usually 0.5)
Relative ETR measurement is achieved by multiplying Y(II) by the irradiation light level in the PAR range (400
nm to 700 nm) in µmol/m2/s, multiplied by the average ratio of light absorbed by the leaf 0.84, and
multiplied by the average ratio of PSII reaction centers to PSI reaction centers, 0.50.
Relative ETR values are used as valuable parametr for stress measurements when comparing one plant to
another, as long as the plants to be compared have similar light absorption characteristics.
ETR measurement in FluorCam (FC)
In FluorCam ETR values are calculated based on the equation ETR = Y(II) × PAR × 0.8 × 0.5.
To measure ETR parametr in FluorCam it is recommended to use Light Curve protocol (Fig. 1) that can be
launched from the Protocol Menu. Light curve protocol for Actinic light 1 and Light Curve protocol for Actinic
light 2 is available.
FM Lss1
FM Lss2
FM Lss3
FM Lss4
FM Lss5
FM Lss6
FM
Ft Lss1
Ft Lss2
Ft Lss3
Ft Lss4
Ft Lss5
Ft Lss6
F0
Saturating pulses
Mf
Actinic light of increasing intensities L1-L6 + Measuring flashes
Fig.1 Schematics of Light curve protocol kinetics.
Light Response Curves are a means of quantifying the rate of photosynthetic performance at different light
irradiances. Light response curves (LRCs) of electron transport rate (ETR), effective quantum yield (Y(II)), and
non-photochemical quenching (NPQ) against light intensity (PPFD) can be used as valuable tool to estimate
the photosynthetic light-use efficiency in response to different stresses.
Light Curve protocol in FC is designed to measure quenching analysis in light adapted state at different light
irradiances (Fig. 2). As a result of the measurement, range of quenching analysis parametres are calculated
for different light intensities such as Fv´/Fm´(Fv/Fm_Lss), QY_Lss (equal to Y(II), Fq´/Fm, φPSII, Genty
parametr,...), NPQ_Lss, qP_Lss, qL_Lss, qN_Lss and ETR_Lss.
In the Protocol Menu of FC Light Curve protocols are pre-defined with duration of actinic light exposure for
60 s at 6 irradiances that are gradually increasing (Fig. 2). The length of the irradiance period (actinic light
exposure) and the number of irradiance steps can be optimised based on user´s needs. The user is adviced to
contact PSI support info@psi.cz if modifications in the protocol are required such as duration of the actinic
light exposure or number of irradiance steps. For example light curve protocols with 12 irradinace steps can
be used for detail characterisation of photosynthetic performance during mutant screen in Arabidopsis
thaliana (Ref. 1).
L6
L5
L4
L3
L2
Increasing
irradiance
L1
Actinic light exposure
Fig.2 Light curve protocol with schematics of actinic light exposure. Irradiance is gradually increasing from L1 to L6 actinic light
exposure step.
In the Light Curve protocol light irradiances are defined as percentage of the maximum light intesity (for
Actinic light 1 or Actinic light 2), which is available for the given hardware of FluorCam configuration (Fig. 3).
The user can use the pre-defined protocol settings for the light irradiance or modify the percentage numbers
according to his/her specific requirements.
Fig.3 Light curve protocol with highlighted commands for the light intensity settings- Light intesity is defined as percentage of the
maximum light intensity (0-100%).
For ETR calculation PAR light irradiance in µmol/m2/s is required. To convert the percentage of light intensity
into light intensity expressed in µmol/m2/s the user will need to perform light calibration or use light
calibration sheet provided by the manufacturer (Fig. 4).
Light intesity for the given percentage of actinic light can be measured with Light Meter (e.g. LI-250A Light
Meter from Li-Cor) or with spectroradiometer SpectraPen LM500.
Please note, that the light calibration should be performed in defined distance from the light source. This
distance should be always the distance, which is between the measured object (plant surface) and the light
source.
Actinic light 2
Intensity
Act 2 [%]
[µmol/m2/s]
Actinic light 2 intensity
0
10
128
20
345
30
563
40
782
50
1001
60
1220
70
1436
80
1650
90
1863
100
2064
2500
Intensity [µmol/m2/s]
0
y = 212.35x - 269.4
R² = 0.999
2000
1500
1000
500
0
0
10
20
30
40
50
60
70
80
90 100
Act 2 [%]
Fig.4 Example of light calibration for Actinic light 2.
Quick Guide for ETR measurement with Light Curve Protocol
1. Select the Light Curve protocol you would like to use (with Act1 or Act2 light exposure) from Protocol
Menu
2. To proceed with the measurement Light Calibration is required for conversion of light intensity
expressed in the percentage into light intensity expressed in µmol/m2/s.
3. Perform the light calibration for the actinic light choosen for the Light Curve protocol, plot in the
graph the light intesity in percentage and equivalent values in µmol/m2/s, add the linear trendline
and calculate the equation (example is shown in fig. 4).
4. Go to Protocols window and insert the equation values from the graph into the protocol, where
variable LightA and Light B is defined. See below and figure 5 for more information.
Example of equation from Fig. 4:
y = 212.35x - 269.4
R² = 0.999
Equation variable defined in the Light Curve protocol are:
LightA=212.35
LightB=-269.4
Fig.5 Light curve prococol with highligted row where equation values from the light calibration curve should be inserted.
5. In the next step it is important to define the light intensity in percentage for the six irradiance steps
the user wants to use for the light response curve measurement and determination of ETR.
6. Define the percentage of the light irradiance in the Light Curve protocol as shown in fig. 7.
Fig.6 Light curve prococol with highligted row where light intensity is defined in percentage for the six irradinace steps.
7. The values in Light intensity row (as shown in fig. 6) must correspond to the values defined in the
protocol body as shown in fig .3 and fig. 7.
8. Store the protocol for later use and measurements.
9. Go to Live window and optimise the camera settings as shutter and sensitivity to obtain suitable
signal for minimal (F0) or instaneous (Ft) fluorescence of the object. Let measuring flashes switched
on and adjust El. Shutter and Sensitivity in LIVE WINDOW. Suitable signal should be in the range of
200-500 digital units (dark blue or blue color of Extended spectrum 3_0_3, which is the most
sensitive color scales for human eye).
10. Let measuring flashes switched on and adjust Actinic light intensity by trucking the bar. Use
maximum light intensity of the desired actinic light used in the Light Curve protocol. Next switch ON
Super in Light Sources panel in LIVE WINDOW and adjust it. Super light intensity should be saturating
even at the highest actinic light intensities, therefore light intesity higher then highest actinic light
intesity should be used. The saturating pulse is only switched for limited time (800ms), so it must be
switched on each time the intensity is changed.
11. Finally, test image quality with all 3 lights (Flashes + Act + Super) switched on as described above.
CCD camera is 12 bits, so fluorescence signal can be measured between 0 – 4096 digits. If the used
settings are saturating for the tested object, the error message PIXELS OVERFLOW at the bottom of
the LIVE WINDOW will warn you. In this case the dynamic range of the camera is exceeded and the
signal above 4096 digits is not integrated. It is necessary to decrease Sensitivity or both Sensitivity +
El. Shutter to reach ideally fluorescence signal of 2000-3000 digits when all light sources (Flashes +
Act at maximum intensity used + Super) are switched ON.
Fig.7 Protocol body with highlighted rows where percentage of light irradiance is defined and must be corresponding.
12. IMPORT the optimized camera and light settings to the protocol by clicking the button Use in the
bottom of LIVE WINDOW.
13. To start the measurement click red flash icon, Start Experiment, in the top panel to run the
measurement.
14. Calculated numeric ETR values for the individual light irrandiances can be after the analysis step
exported, further processed and used to generate graph for the light response curves of electron
transport rate (ETR) as shown in fig. 8.
40
35
30
ETR
25
20
15
10
5
0
0
500
1000
1500
2000
PAR (umol/m2/s)
Fig.8 Light response curves of electron transport rate (ETR).
References
1. Chen KM, Holmström M, Raksajit W, Suorsa M, Piippo M, Aro EM.(2010). Small chloroplast-targeted DnaJ
proteins are involved in optimization of photosynthetic reactions in Arabidopsis thaliana. BMC Plant Biol.
2010 Mar 7;10:43. doi: 10.1186/1471-2229-10-43.
2. Zhang X, Wollenweber B, Jiang D, Liu F, Zhao J.(2008). Water deficits and heat shock effects on
photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription
factor. J Exp Bot. 2008;59(4):839-48. doi: 10.1093/jxb/erm364. Epub 2008 Feb 13.
05/2015
© PSI (Photon Systems Instruments), spol. s.r.o.