Leaf total chlorophyll content and photochemical efficiency (Fv/Fm) are physiological parameters commonly used as indicators of leaf senescence. Total chlorophyll content was measured by extracting approximately 0.1 g of leaves in 10 mL in 80% acetone in the dark for 24 h and measuring the absorbance of extracts at 663 nm and 645 nm using a spectrophotometer as described earlier [5 (link),6 (link)]. Fv/Fm was determined non-invasively after leaves were adapted to dark conditions for 30 min. A chlorophyll fluorometer (Imaging-PAM-M series, Heinz Walz GmbH, Effeltrich, Germany) equipped with a charge-coupled device (CCD) camera to capture high-resolution digital images of the emitted fluorescence from the dark-adapted leaves.
Imaging pam m series
Manufactured by Walz
Sourced in Germany
The IMAGING-PAM M-Series is a compact and versatile fluorescence imaging system designed for non-invasive analysis of photosynthetic processes in plants and algae. It provides high-resolution images of chlorophyll fluorescence parameters, enabling the assessment of photosynthetic efficiency and stress responses in a variety of samples.
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Lab products found in correlation
Fluar 40 1.3 na objective, by Zeiss (3 mentions)
Lumar v12, by Zeiss (2 mentions)
Axioscope a1 epifluorescence microscope, by Zeiss (2 mentions)
Ccd camera imag k6, by Zeiss (2 mentions)
Dual pam 100, by Walz (1 mentions)
Gsf 3000, by Walz (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Imag k6, by Zeiss (1 mentions)
20 protocols using imaging pam m series
1
Quantifying Leaf Chlorophyll and Photochemistry
2
Photosynthetic Imaging of N. benthamiana
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Video imaging was performed on detached N. benthamiana leaves with the IMAGING‐PAM M‐Series (Heinz Walz, Effeltrich, Germany) using 15 min of blue actinic light (600 μmol photons m−2 sec−1), followed by 10 min of dark recovery. Unless otherwise stated, samples were dark‐acclimated for 20 min prior to measurements. Photosynthetic parameters were calculated as described (Brooks and Niyogi, 2011 ).
The Dual‐PAM‐100 (Heinz Walz) was used to measure chlorophyll a fluorescence of N. benthamiana leaf discs expressing various constructs involved in NPQ and carotenoid biosynthesis. Unless otherwise indicated, samples were dark‐acclimated for 20 min prior to all treatments. Samples were treated with 10 min of red actinic light (660 μmol photons m−2 sec−1) followed by 10 min of dark recovery.
The Dual‐PAM‐100 (Heinz Walz) was used to measure chlorophyll a fluorescence of N. benthamiana leaf discs expressing various constructs involved in NPQ and carotenoid biosynthesis. Unless otherwise indicated, samples were dark‐acclimated for 20 min prior to all treatments. Samples were treated with 10 min of red actinic light (660 μmol photons m−2 sec−1) followed by 10 min of dark recovery.
3
Chlorophyll Fluorescence Analysis of Azolla
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The chlorophyll fluorescence parameters were measured using an Imaging Pam M‐series fluorimeter (Heinz Walz GmbH). Before starting the measurements, plants were adapted to dark conditions for 30 min, and maximal PSII quantum yield (Fv/Fm) was calculated after applying a saturating pulse, according to Genty et al. (1989 ). Then, actinic light was stepwise increased to 11, 21, 26, 56, 81, 111, 146, 186, 231, 281, 336, 461, 531, 611, and 700 μmol m−2 s−1 and values of electron transport rate (ETR), effective PSII quantum yield (ΦPSII), non‐photochemical quenching (NPQ), and the coefficient of non‐photochemical quenching (qL, indicating the fraction of open PSII reaction centers) were calculated according to Genty et al. (1989 ), Maxwell and Johnson (2000 (link)), and Baker (2008 ). All chlorophyll fluorescence parameters were measured in different points (10–62) simultaneously in a single A. filiculoides sample and pooled with those collected in different samples (3–5) of A. filiculoides grown within the same batch under the same light intensity to have mean representative values ± se for each light condition.
4
Chlorophyll Fluorescence Analysis under Heat Stress
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The heat-induced changes of chlorophyll a fluorescence parameters were also detected on intact detached leaves by the use of a pulse amplitude modulated fluorometer (Imaging-PAM M series, Walz, Effeltrich, Germany) completed with a thermoregulatory instrument consisting of a water-cooled Peltier thermoelectric module, a thermocouple thermometer, and a control unit. The measurements were started at 21°C and after the photosynthesis was steady (15 min) under actinic light illumination at 100 µmol m−2 s−1 the temperature was increased from 21 to 55°C at a rate of 1°C min−1. During the measurements, 1.0 s saturated flashes (photosynthetic photon flux density = 3,000 µmol m−2 s−1) provided by an LED-Array Illumination Unit IMAG-MAX/L (λ = 450 nm) were applied at each degree Celsius. The effective quantum yield of PS (II) parameter was shown.
5
Leaf Senescence Physiological Indicators
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Leaf total chlorophyll content and Fv/Fm are physiological parameters commonly used as indicators of leaf senescence. Total chlorophyll content was measured by extracting approximately 0.1 g of leaves in 10 mL in 80% acetone in the dark for 24 h and measuring the absorbance of extracts at 663 nm and 645 nm using a spectrophotometer as described earlier [31 (link)]. Fv/Fm was determined noninvasively after leaves were adapted to dark conditions for 30 min through a chlorophyll fluorometer (Imaging-PAM-M series, Heinz Walz GmbH, Effeltrich, Germany) equipped with a charge-coupled device (CCD) camera that enabled the capture of high resolution digital images of the emitted fluorescence.
6
Chloroplastic ETR Measurement Using IMAGING-PAM
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Chloroplastic ETR measurements were performed using an IMAGING-PAM M-Series (MAXI version) (Walz; Effeltrich, Germany). ETR response curves were obtained using an inbuilt script (settings: 2, corresponding to the following PAR: 0, 1, 11, 21, 36, 56, 81, 111, 146, 186, 231, 281, 336, 396, 461, 531, 611, 701, 801, 926 , 1,076-µmol photons m -2 s -1 , 20 s interval between each measurement). The ETR of technical replicates (6-12 areas of interest) was combined to get the leaf ETR, and the ETR of different leaves for the same treatment were plotted to get the treatment ETR trend. A Loess regression was adjusted to each curve with a 95% confidence interval using the R package ggplot2.
7
Chlorophyll a Fluorescence Measurement
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For chlorophyll a fluorescence analyses, seedlings were dark-adapted for 15 min prior to PAM measurements, using the IMAGING-PAM M-Series (Heinz Walz GmbH, Effeltrich, Germany). The following settings were used for data collection: Gain = 1, Set Damping = 2, Measure Light = 2, Means Freq = 1. The following program was used for measurement of plants: Fv/Fm estimation, pause for 5 s, actinic light set at 56 PAR for 10 min and measurement (low light; LL), followed by actinic light set at 461 PAR for 10 min and a final measurement (high light; HL).
8
Measuring Photosynthetic Efficiency in Arabidopsis
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The FI analysis was carried out using pulse amplitude modulated fluorometer (PAM) with a blue LED-Array Illumination Unit IMAG-MAX/L (λ = 450 nm) (Imaging-PAM M-Series, Walz, Effeltrich, Germany) on the fully expanded leaves of Arabidopsis plants, which were exposed to dark for 15 min in order to reach the open state of the acceptor side of the electron transport chain. The determination of the maximum quantum efficiency (Fv/Fm) and the actual quantum yield [Y(II)] of photosystem 2 was carried out as it was described in [39 (link)].
9
Photosynthesis and Chlorophyll Fluorescence
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The photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 (Ci) and water use efficiency (WUE) of leaves were measured using a potable photosynthesis system (GSF-3000, Heinz-Walz Instruments, Effeltrich, Germany). Intact leaves were measured at a temperature of 25°C, a photosynthetically active radiation (PAR) of 750 μmol m-2 s-1, a CO2 concentration of 400 μmol mol-1 and relative humidity between 40 and 60%.
A modulated imaging chlorophyll fluorometer (IMAGING-PAM M-Series, Heinz-Walz Instruments, Effeltrich, Germany) was used to measure chlorophyll fluorescence parameters according to the instructions provided by the manufacturer. After dark adaptation for 30 min, the fluorescence (F0), maximum fluorescence (Fm) and the non-photochemical quenching (NPQ) were determined (Zhou et al., 2010 (link)). The maximal quantum yield of PSII photochemistry in the dark-adapted state was calculated as Fv/Fm.
A modulated imaging chlorophyll fluorometer (IMAGING-PAM M-Series, Heinz-Walz Instruments, Effeltrich, Germany) was used to measure chlorophyll fluorescence parameters according to the instructions provided by the manufacturer. After dark adaptation for 30 min, the fluorescence (F0), maximum fluorescence (Fm) and the non-photochemical quenching (NPQ) were determined (Zhou et al., 2010 (link)). The maximal quantum yield of PSII photochemistry in the dark-adapted state was calculated as Fv/Fm.
10
Determining Photosynthetic Efficiency in Plants
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To determine maximum quantum efficiency of photosystem II photochemistry (Fv/Fm), sterile and stratified seeds were sown on 1/2 Murashige & Skoog plates containing 8 g L−1 agar (pH 5.8). After 14 d, seedlings were transferred to liquid culture medium. Plants were cultivated on full nutrient solution for another 14 d and then transferred to either control or Mn-free conditions for 10 d. Photosynthetic parameters were determined by PAM fluorescence imaging (IMAGING-PAM M-Series, Walz). Before measurements, plants were dark-adapted for 30 min. Images were captured, and saturating flashes (902 μmol m−2 s−1) were used to measure Fv/Fm.
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