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Pulse amplitude modulated (PAM) measurement of variable fluorescence is a technique that allows quantification of effective quantum yields of photosystem II (qPSII), coefficients of photochemical (qP) and non-photochemical (qNP) quenching, and other parameters used in photosynthesis research of plants and cyanobacteria. The Imaging-PAM system by Walz GmbH allows these parameters to be measured as images.
The ImagingWin software provided by Walz for the PAM-imaging measurements makes it possible to do some basic data analyses. For example, one can select a region of interest in the image and the software displays the corresponding time-series of the acquired or processed data such as fluorescence yields, qPSII, qP, qNP, etc.
To characterize light acclimation of a photosynthetic organism, the software makes it possible to measure so-called light curves (LC), which is a sequence of fluorescence yields measured at increasing intensities of the actinic light (F') accompanied by fluorescence yields measured during a saturating pulse at each actinic light level (Fm'). Based on such measurements, effective quantum yields of photosystem II can be calculated as qPSII = (Fm'-F')/Fm' at each actinic light level, from which the light acclimation intensity (I_acc) can be derived.
Although the ImagingWin software makes it possible to calculate I_acc in a given region of interest, it does not allow the calculation of I_acc for the entire image. Such and other calculations are possible with the Look@PAM software.
To make use of the Look@PAM software, one needs to measure the LC images of F' and Fm' together with the reflectance images under red and near-infrared illumination, and export the entire image set as a multi-page TIF image. This multipage TIF image can then be directly loaded with Look@PAM and analyzed. Also, for calibration purposes, one needs to have a tif image from the fluorescence standard, which will be used to correct for the possibly uneven illumination of the imaged field.
The Look@PAM program is written in Matlab. To run it you need to have Matlab installed on your system. Then, all you need to do is to download the Matlab source code of Look@PAM from here, unpack it to a directory of your choice and run
lookatpam from the Matlab's command window. Before you do this, edit the
input_parameters.m files to set some useful global settings.
Unfortunately, I did not have time to write a manual for the program. But by using the program's menu it should be fairly simple to process the data. If it is not the case, please send me an email to ask questions about what is not clear.