The chlorophyll-a fluorescence was measured by using a pulse amplitude modulated fluorometer (Imaging-PAM M-Series fluorometer; Walz, Effeltrich, Germany). The maximum quantum yield of PSII photochemistry Fv/Fm was measured on 20 min dark-adapted leaves. Fv/Fm= (Fm-F0)/Fm, where Fm is the maximum fluorescence induced by a saturating flash (8000 μmol m−2 s−1 PPFD for 0.8s) in dark adapted leaves, F0 is the minimum chlorophyll fluorescence yield in the dark (PPFD < 1 μmol m−2 s−1). The effective PSII quantum yield (YII) was measured at a light intensity of 250 μmol m−2 s−1 and represents the proportion of absorbed light energy being used in photochemistry. It is calculated as: (Fm’−F)/Fm’, Fm’ is the maximum fluorescence level induced by a saturating light pulse at the steady state, and F is the steady state chlorophyll fluorescence immediately prior to the flash.
Imaging pam m series fluorometer
Manufactured by Walz
Sourced in Germany
The Imaging-PAM M-Series fluorometer is a versatile instrument designed for chlorophyll fluorescence measurements. It provides non-invasive and quantitative analysis of photosynthetic performance in various sample types, including leaves, algae, and cyanobacteria.
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7 protocols using imaging pam m series fluorometer
1
Chlorophyll Fluorescence Analysis
2
Chlorophyll-a Fluorescence Quenching Analysis
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Chlorophyll-a fluorescence quenching analysis was carried out using a pulse amplitude modulated fluorometer (Imaging-PAM M-Series fluorometer; Walz, Effeltrich, Germany). The Fv/Fm parameter was determined on plants previously dark-adapted for 20 minutes, using a 0.8 s saturating pulse (PPFD = 3000 μmol m-2 s-1) provided by a LED-Array Illumination Unit IMAG-MAX/L (λ = 450 nm). Photosynthesis was then activated using 230 μmol m-2 s-1 actinic light intensity for 15 min and quenching analysis was performed using 0.8 s saturating pulses applied every 30 s. The quenching parameters were determined under steady state conditions according to the nomenclature described by Klughammer and Schreiber [18 ].
3
Chlorophyll Fluorescence Imaging Protocol
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Chlorophyll fluorescence of inoculated leaves was measured using an IMAGING-PAM M-series fluorometer (MAXI-version, Heinz Walz GmbH, Effeltrich, Germany) according to the manufacturer’s manual (see supplemental methods 1 for details).
4
Calculating Photosynthetic Electron Transport in Lichens
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The electron transport rate (ETR) = ΦPSII × PAR × 0.5 × Abs (Baker 2008 (link)) ΦPSII = effective quantum yield of PSII; 0.5 assumes equal absorption of photons in PSII and PSI; Abs = fraction of incident light absorbed in PSII and PSI). ΦPSII was measured from 0 to 450 μmol photons m−2 s−1 in late summer, and from 0 to 1250 μmol photons m−2 s−1 in spring samples, using a red light ImagingPAM M-series fluorometer (Heinz Walz GmbH, Effeltrich, Germany). The Abs parameter is assumed to be 0.85 in green leaves, but is hard to estimate in lichens in which it is lower due to screening pigments (Solhaug et al. 2010 (link)). We assessed apparent ETR (ETRApp) setting Abs = 1. Because ETRApp does not include the unknown Abs parameter, it is higher than the real ETR. Because some fluorescence also comes from lower parts of the lichen canopy resulting in higher ΦPSII, ETR will be overestimated. For C3 plants, the ratio between ETR and photosynthetic gross CO2 uptake (ETR / CO2gross) is on average between 7.5 and 10.5 (Perera-Castro and Flexas 2023 (link)).
5
Chlorophyll Fluorescence Imaging of Chlamydomonas Cells
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A MAXI-version of the pulse-modulated Imaging-PAM M-series fluorometer (Heinz Walz GmbH, Effeltrich, Germany) was used to determine the Chl fluorescence imaging [44 (link)]. Before measurements, samples were adapted for 0.5 h in the dark under the imaging system camera after focusing the camera. Under 0.25 μmol (photons) m−2 s−1 measuring light, 110 μmol (photons) m−2 s−1 actinic light and 6000 μmol (photons) m−2 s−1 saturation pulse light, fluorescence images were monitored. The following parameters were measured directly: FV/FM and ETR.
For the measurement of C. reinhardtii cells, 200 μL of suspensions (A750 of about 0.65) with 10% acetone (mock) and CA were added into the 96-well black microtiter plate to a final CA concentration of 20, 30 and 40 μM, respectively. The treated cells were incubated for 2.5 h at 25 °C under 100 μmol (photons) m−2 s−1 white light, and then samples were adapted in darkness for 0.5 h before fluorescence determination.
For the measurement of C. reinhardtii cells, 200 μL of suspensions (A750 of about 0.65) with 10% acetone (mock) and CA were added into the 96-well black microtiter plate to a final CA concentration of 20, 30 and 40 μM, respectively. The treated cells were incubated for 2.5 h at 25 °C under 100 μmol (photons) m−2 s−1 white light, and then samples were adapted in darkness for 0.5 h before fluorescence determination.
6
Chlorophyll Fluorescence Quenching in Wheat
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Chlorophyll a fluorescence quenching analysis was carried out using a pulse amplitude modulated fluorometer (Imaging-PAM M-Series fluorometer; Walz, Effeltrich, Germany) on detached leaves of wheat plants with or without salt stress (500 mM NaCl) for 5 days. The plants were previously dark-adapted for 20 minutes, after which the F v /F m parameter was determined using a 0.8 s saturation pulse (PPFD=3000 µmol m -2 s -1 ) provided by a LED-Array Illumination Unit IMAG-MAX/L (λ=450 nm). Photosynthesis was then activated using 230 µmol m -2 s -1 actinic light intensity for 15 min and the quenching analysis was performed using a 30 s saturation pulse frequency. The quenching parameters were determined under steady state conditions according to the nomenclature described by Klughammer and Schreiber (2008) .
7
Chlorophyll Fluorescence Measurements in Plants
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Chlorophyll-a fluorescence was measured using a pulse amplitude modulated fluorometer (Imaging-PAM M-Series fluorometer; Walz, Effeltrich, Germany). The maximum quantum yield of PSII photochemistry, F v /F m , as F v /F m = (F m -F 0 )/F m , where F m is the maximal fluorescence induced by a saturating flash (8000 µmol m -2 s -1 PPFD for 0.8 s) in leaves dark-adapted for 20 min, and F 0 is the minimum chlorophyll fluorescence yield in the dark (PPFD < 1 µmol m -2 s -1 ). (As no significant changes were observed in the F v /F m parameter, these data are not shown.) The effective PSII quantum yield (∆F/Fm') which represents the proportion of absorbed light energy consumed in photochemistry, and was measured at a light intensity of 250 µmol m -2 s -1 calculated as (Fm'-F)/Fm', where Fm' is the maximal fluorescence level induced by a saturating light pulse in the steady state, and F is the steady state chlorophyll fluorescence immediately prior to the flash. Measurements were performed on the last fully expanded leaves.
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