300412 UE Basic Methods in Plant Physiology, Anatomy and Ecology
Measuring the electron transport rate with the
Imaging PAM
Description:
• Chlorophyll fluorescence is a very sensitive indicator of photosynthesis. Quantitative information on the
quantum yield of photosynthetic energy conversion is obtained by PAM fluorometry and the saturation
pulse method (Schreiber U (2004) Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse
Method: An Overview, pp. 279-319. Kluwer Academic Publishers, Dordrecht, The Netherlands).
• A wide range of photosynthetic parameters can be derived from fluorescence measurements, giving insight into the physiological state of all photosynthetically active organisms, including higher plants, mosses
and ferns as well as various types of algae, phytoplankton and biofilms.
• With the advance of highly sensitive CCD cameras and extremely strong light emitting diodes (LED) development of IMAGING-PAM fluorometers has become possible that not only measure images of chlorophyll
fluorescence but are also fully competent in providing all relevant chlorophyll fluorescence parameters
using the saturation pulse method. In this way, images of photosynthetic activity and its spatio-temporal
variations can be obtained.
• All IMAGING-PAM fluorometers provide images for 17 different parameters. The fluorescence parameter
Ft is continuously monitored. Fo and Fm are assessed after dark adaptation, serving as reference for fluorescence quenching analysis by the saturation pulse method.
• Besides Fv/Fm, the PS II quantum yield after dark acclimation, also the PS II quantum yield during illumination, Y(II), and the quantum yields of regulated and non-regulated energy dissipation, Y(NPQ), Y(NO) as
well as the apparent electron transport rate (ETR and PS) can be imaged.
• A routine for measuring a PAR-absorptivity image is provided for the MAXI and the MINI version of the
IMAGING-PAM (Abs.-image based on images of NIR and red light remission). From this parameter also the
apparent rate of photosynthesis is calculated and does not need to use the commonly known PAR-absorptivity mean value of 0.84 (ETR) for all areas anymore.
• The parameter PS/50 is displayed in order to depict the apparent photosynthesis with the help of the false
color code that is also used for the other photosynthesis parameters.
Chlorophyllfluorescence - Imaging PAM
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Preparation of the instrument
1. Connect al the cables. Don’t forget the firewire connection of the camera.
2. Swith on the PC/Labtop and the Imagning PAM
3. Start ImagingWin.exe
Choose the following
MINI is our System and then
Settings:
Choose TAB Settings and Reset Defaut - Settings (near the bottom right).
ETR--measurement
Tab Light curve
EDIT and DEFAULT settings (each 10 seconds the light intensity is increased from 0 to 726 µmol m-2 s-1.
A “0” at a certain light intensity (PAR) stoppt the lightcurve
A better scaling in achieved with the option Ruler.
Choose Kinetics -7 Fm’, F, Y(ll).
A report show all the recorded data. Is it possible to all the calculated values like Fm, FO, ETR, etc.
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Chlorophyllfluorescence - Imaging PAM
Measurement:
1. Insert a leaf in a flat position.
2. Image: the leaf is displayed. If not, select Ft and Measure (Ft is the actual fluorescence). Be careful, that
the actual leaf is displayed and not the the leaf of the last measurement.
3. With Image you shlould choose measurement areas: AOI (area of interests) on the right side.
It is possible to choose different shapes of the AOI’s. To close the AOI’s shape the start and the end of the encircling line have to be connected. The actual fluorecence value and the appropriate number will be displayed.
With Delete you can erase a AOI.
Even after the Measurement AOI can be added, because the measurement captured the whole leaf.
4. Light Curve ⇨ ETR
5. START
6. To display the values of an AOI, open the AOI.
Regardless of an an error occurs, the measurement is fulfilled.
The ETR-measurement is finished after aprox. 240 sec.
7. The curves should be stored (*.pim-files). Press the disk-symbol left-bottom. This file contains all
relevant data.
8. The report-files can be importet into a spred-sheet program like Excel). To to this the report-files
have to be stored in the *.csv-formet (value are comma separated).
Report (symbol with the red arrow at the top under the menu with Image, light curve, ..) ⇨ the file has the
same name but the extension *.csv.
9. Start are new measurement like under point 1. and 2. A “NO” is nessesary to erase the memory.
10. The question, if the Fo and Fm-values should be retained ⇨ NO.
Chlorophyllfluorescence - Imaging PAM
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