Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research

We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chl a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves.     

银杏叶萜内酯含量的变化及其与叶绿素荧光特性的关系

为探究银杏(Ginkgo biloba)叶萜类内酯含量和光合同化作用的关系,对其内酯含量和叶绿素荧光特性进行了研究。结果表明,不同采收时间银杏叶中白果内酯和银杏内酯含量有显著差异,总体上,5月份含量较低,此后逐渐升高,8月份达到高峰,然后快速下降,10月底最低;与此同时,银杏叶片的光合色素以及叶绿素荧光参数也呈现周期性变化。白果内酯以及萜内酯含量与叶绿素荧光参数Y(NPQ)之间呈极显著正相关关系,因此,可以通过银杏叶片的叶绿素荧光参数预测白果内酯和萜内酯含量。     

An instrument capable of imaging chlorophyll a fluorescence from intact leaves at very low irradiance and at cellular and subcellular levels of organization

An instrument capable of imaging chlorophyll a fluorescence, from intact leaves, and generating images of widely used fluorescence parameters is described. This instrument, which is based around a fluorescence microscope and a Peltier-cooled charge-coupled device (CCD) camera, differs from those described previously in two important ways. First, the instrument has a large dynamic range and is capable of generating images of chlorophyll a fluorescence at levels of incident irradiance as low as 0.1 μmol m^?2 s^?1. Secondly, chlorophyll fluorescence, and consequently photosynthetic performance, can be resolved down to the level of individual cells and chloroplasts. Control of the instrument, as well as image capture, manipulation, analysis and presentation, are executed through an integrated computer application, developed specifically for the task. Possible applications for this instrument include detection of early and differential responses to environmental stimuli, including various types of stress. Images illustrating the instrument's capabilities are presented.     

Definition and Evaluation of the Spatio‐Temporal Variations in Chlorophyll Fluorescence during the Phases of CAM and during Endogenous Rhythms in Continuous Light,in Thick Leaves of Kalanchoë daigremontiana5

Abstract: We used chlorophyll fluorescence imaging to examine the homogeneity of photosynthetic metabolism during CAM in the thick leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Intense, persistent fluorescence from a DCMU treated thin leaf of Clematis sp placed beneath a K. daigremontiana leaf was readily detected through the thick leaf. Evidently reabsorption of fluorescence was qualitatively unimportant in the system used. Chlorophyll fluorescence images from 7 mm tissue discs excised from Kalanchoë leaves were collected at 60 s intervals during 20 min transients elicited by red excitation light. Information about patchiness and subsurface processes was gained by statistical factor analysis and Fourier transform. Although small, highly resolved rings of bright chlorophyll fluorescence surrounding discs of low fluorescence were observed from cells near the surface, no independent regional temporal variation in fluorescence was evident in the surface‐biased images. Temporally independent chlorophyll fluorescence was present in images biased towards sub‐epidermal sources, in most phases of CAM, and during endogenous rhythm. These asynchronous changes were several millimetres apart. This patchy fluorescence was confirmed when attached leaves were excited with blue light in a leaf chamber while CO₂ and H₂O exchange was monitored. Large spatio‐temporal variations in the efficiency of photosystem II were always observed during phases II and IV of CAM, when both CO₂ fixation cycles are active, and during the maximum rate of CO₂ fixation during the endogenous rhythm in continuous light. These data are discussed in terms of metabolic isolation in the thick but uniform tissues in which gas diffusion may be largely confined to wet cell walls, thereby rendering the tissue functionally heterobaric. Prolonged, but in some instances, reversible alterations in PSII efficiency could be produced by injection of metabolic inhibitors, confirming that patchy fluorescence may reflect the differing energy costs of photosynthesis in different CAM phases.     

Measurement of Differences in Red Chlorophyll Fluorescence and Photosynthetic Activity between Sun and Shade Leaves by Fluorescence Imaging

With a flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) the photosynthetic activity of several thousand image points of intact shade and sun leaves of beech were screened in a non-destructive way within a few seconds. The photosynthetic activity was determined via imaging the Chl fluorescence at maximum F_p and steady state fluorescence F_s of the induction kinetics (Kautsky effect) and by a subsequent determination of the images of the fluorescence decrease ratio R_Fd and the ratio F_p/F_s. Both fluorescence ratios are linearly correlated to the photosynthetic CO₂ fixation rates. This imaging method permitted to detect the gradients in photosynthetic capacity and the patchiness of photosynthetic quantum conversion across the leaf. Sun leaves of beech showed a higher photosynthetic capacity and differential pigment ratios (Chl a/b and Chls/carotenoids) than shade leaves. Profile analysis and histogram of the Chl fluorescence yield and the Chl fluorescence ratios allow to quantify the differences in photosynthetic activity between different leaf parts and between sun and shade leaves with a high statistical significance.     

Structural and functional characteristics of photosynthetic apparatus of chlorophyll-containing grape vine tissue

Photosynthesis in tissues under periderm of woody stems and shoots of perennial plants occurs in environment that is very different from the internal environment of leaf chloroplasts. These tissues are characterized by high CO₂ and low O₂ concentrations, more acidic surroundings, besides that only light which have passed through periderm reaches photosynthetic antennas. In contrast to leaves of deciduous plants chlorenchyma tissues of wintering plant organs are exposed to temperature fluctuations during all seasons, that is why the photosynthetic apparatus of woody stems has to be able to adapt to a wide range of environmental temperatures. In order to reveal unique features, which enable photosynthetic apparatus of chlorenchyma cells in woody plant organs to implement biological functions under different light and temperature conditions, we studied photosynthetic tissues of stem cortex in grapevine (Vitis vinifera L.) under normal conditions and after exposure to suboptimal temperatures and high light intensity. Comparative analysis of photosynthetic pigment composition and low-temperature chlorophyll fluorescence emission spectrum of leaves, young shoots and chlorenchyma of lignified shoots revealed relatively high level of chlorophyll b and carotenoids, and high photosystem II (PSII) to photosystem I (PSI) ratio in woody shoots. Analysis of parameters of variable chlorophyll fluorescence revealed high PSII activity in grapevine shoot cortex and demonstrated improved freeze tolerance and higher sensitivity to light of photosynthetic apparatus in grape vine in comparison to leaves. It was shown for the first time that photosynthetic apparatus in chlorenchyma cells of vine undergoes so-called “state-transition”–fast rearrangements leading to redistribution of energy between photosystems. Analysis of fatty acid (FA) compositions of lipids in examined tissues showed that the FA unsaturation index in green tissue of vine is lower than in leaves. A distinct feature of FA compositions of lipids in vine cortex was relatively high level of linoleic acid.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, R_Fd (defined as Chl fluorescence decrease F_d from maximum to steady state fluorescence F_s:F_d/F_s), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio R_Fd is linearly correlated to the net photosynthetic CO₂ fixation rate P _N as is indicated here for sun and shade leaves of various trees that considerably differ in their P _N. Imaging the R_Fd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F₆₉₀. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F₄₄₀/F₆₉₀, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F₆₉₀/F₇₄₀.     

Oscillations in photosynthetic carbon assimilation and chlorophyll fluorescence are different in Amaranthus caudatus,a C4 plant,and Spinacia oleracea,a C3 plant

The characteristics of oscillations in photosynthetic carbon fixation and chlorophyll fluorescence in leaves of the C₄ plant Amaranthus caudatus L. were compared to those shown by the C₃ plant Spinacia oleracea L. As in spinach, oscillations could be observed in Amaranthus when leaves were illuminated after periods of darkening, particularly at temperatures below 20°C, less so or not at all at higher temperatures. However, in contrast to spinach, pronounced oscillations occurred in Amaranthus after a sudden dark/light transition only at low, not at high photon flux densities. Whereas in spinach maxima in carbon uptake were observed slightly after minima in chlorophyll fluorescence had occurred, in Amaranthus maxima in carbon uptake were close to maxima in chlorophyll fluorescence. Since the quantum efficiency of electron transport through photosystem II of the chloroplast electron-transport chain was higher during the minima of chlorophyll fluorescence than during the maxima, the observations suggest that in Amaranthus photosynthetic water oxidation did not occur as synchronously with carbon uptake as in spinach. It is proposed that, in contrast to spinach, photosynthetic oscillations in Amaranthus are related to the diffusional transport of photosynthetic intermediates between mesophyll and bundle-sheath cells.Abbreviations F_o, F_m, F_s initial, maximal and steady-state chlorophyll a fluorescence - PFD photon flux density - Q_A primary quinone acceptor of PSII We are grateful to Professors D.A. Walker, FRS, Robert Hill Institute, University of Sheffield, Sheffield, UK., and Agu Laisk, Chair of Plant Physiology, University of Tartu, Tartu, Estonia, for helpful discussions and to Ms. S. Neimanis for help with the experiments. Our work was performed within the research of the Sonderforschungsbereich 251 of the University of Würzburg. It was supported by the Stiftung Volkswagenwerk. A.S.R. acknowledges also support by the Alexander-von-Humboldt-Stiftung and U.G. by the Graduate College of the University of Würzburg.     

Dichlorophenyl dimethylurea (DCMU) induced increase in chlorophyll a fluorescence intensity – An index of photosynthetic oxygen evolution in leaves, chloroplasts and algae

Dichlorophenyl dimethylurea (DCMU) treatment in photosynthetic samples resulted in an increase in the level of steady state chlorophyll a fluorescence at room temperature which was directly proportional to the photosynthetic efficiency. The applicability of this method for the rapid determination of the efficiency of oxygen evolution in leaves, algae, mesophyll cells and chloroplasts has been investigated. Especially reliable values with less than 5% error were obtained if the fluorescence measurements were made under low excitation intensities with a sample chlorophyll concentration below 1.0 μg/ml.     

Leaf and photosynthetic characteristics of pioneer and forest species in tropical montane habitats

Gas exchange and chlorophyll a fluorescence measurements of expanding and adult leaves of four plant species were compared under field conditions. The pioneer species (PS) tended to have thinner leaves with lower nitrogen content and higher stomatal density compared to forest species (FS). Expanding leaves featured lower photosynthetic pigment contents and gas exchange capacity than adult leaves consistent with an immature photosynthetic apparatus. At the time of maximum irradiance, sun-exposed leaves of both PS and FS showed alteration of initial, variable, and maximum fluorescence as well as their ratios indicating photoinhibition. However, leaves recovered to some extent at predawn, suggesting the activation of photoprotective mechanisms. Sun-exposed leaves had comparable responses to high irradiance.     

Physiological characterization of photosynthesis,chloroplast ultrastructure,and nutrient content in bracts and rosette leaves from <Emphasis Type="Italic">Glaucium flavum</Emphasis>

Glaucium flavum is a biennial plant that bears a rosette of leaves, producing a flower stalk, bracteate monochasium, in its second year. The aims of this work were both to investigate the contribution of bracts to gas-exchange activities in this species and to compare this contribution to that of rosette leaves. In addition, we investigated the extent to which its responses can be explained by chloroplast ultrastructure, as well as the possible role of nutrient concentrations in the physiological responses of both leaf types. Gas exchange and plant characteristics regarding chlorophyll fluorescence were examined in a field experiment; we also determined leaf relative water content, tissue concentrations of photosynthetic pigments, chloroplast ultrastructure and nutrient contents. Although bracts indeed contributed to gas-exchange activities of G. flavum, rosette leaves showed higher values of net photosynthetic rate and stomatal conductance to CO₂ for photosynthetic photon flux density above 200 μmol m⁻² s⁻¹. The incongruities in photosynthetic rates between bracts and leaves may be explained by the bigger chloroplasts of rosette leaves, which results in a larger membrane surface area. This agrees with the higher pigment concentrations and quantum efficiency of photosystem II values recorded as well for rosette leaves. On the other hand, bracts showed higher sodium concentrations, which could be a mechanism for salt tolerance of G. flavum.     

Pictorial Demonstrations of Photosynthesis

Theodor Engelmann's experiments in 1882 provided the first recorded visual demonstration of light wavelengths that are absorbed by photosynthetic pigments. Later, starch images in intact leaves were used to demonstrate photosynthesis in green plants. Similarly, light-induced chloroplast movements can form images in leaves as a result of changes in light transmittance through leaves and photoinhibition can form images that can be visualized by whole leaf chlorophyll fluorescence. This paper provides a brief account of how photosynthesis has been used to create an assortment of 'living images' that offer stunning demonstrations of various aspects of photosynthesis.     

基于叶绿素荧光参数的小麦叶片叶绿素相对含量估算方法

叶绿素含量是植物学和农业相关研究领域常用的生理指标.叶绿素含量和叶片光合功能密切相关,但是现有的叶绿素含量的测定方法无法实现叶绿素含量和光合功能的同步测定和关联分析.为解决该问题,本研究通过测定35个小麦品种旗叶的SPAD值和叶绿素荧光诱导动力学曲线,分别使用不同时间的快速叶绿素荧光动力学曲线的荧光值,以及33个常用荧...     

Imaging chlorophyll a fluorescence reveals specific spatial distributions under different stress conditions

Chlorophyll a fluorescence has been adopted as a fast, non-invasive, and cheap method to detect stress effects in plants. The majority of these chl-fluorescence measurements have been carried out with ‘clamping’ fluorometers recording punctual chlorophyll a fluorescence at isolated parts of the leaf. However, this method is inherently limited in providing information on the homogeneity of responses to stresses at the leaf or whole plant level. Therefore the purpose of this study was to measure imaging chlorophyll a fluorescence and to compare the temporal and spatial distribution of this emission under allelochemical (2-3H-benzoxazolinone and 3,4-dihydroxybenzaldehyde), thermal and salt, and heavy metal (cadmium, copper and zinc) treatment in the model plant Arabidopsis thaliana (L.) Heynh. The results suggested different spatial distributions for each condition: the two allelochemicals showed inhibition spots at the edges of the oldest leaves and both did not affect the photosynthetic activity of young leaves; treatment with the three heavy metals revealed highly homogenous effects over the whole plant with a quite uniform decrease of maximum PSII efficiency (also in youngest leaves). On the contrary, temperature (heat and cold) and salt stress showed an initial decrease of fluorescence in the tissues around the vascular bundles that lasted between 2 and 3 h depending on the treatment. These irregularities in chlorophyll fluorescence make it difficult to correlate punctual measures (typical for clamping fluorometers) with the effect on the whole plant, ignoring effects that are evident when imaging is used. Therefore these results show that monitoring chlorophyll a fluorescence by imaging improves the measurement of stress effects on treated plants, suggesting that punctual fluorescence measurements do not always reveal the heterogeneity of the stress-related effects in treated plants.     

Kinetic imaging of chlorophyll fluorescence using modulated light

Fluorometers that measure the kinetics of chlorophyll fluorescence have become invaluable tools for determining the photosynthetic performance of plants. Many of these instruments use high frequency modulated light to measure the rate, efficiency and regulation of photosynthesis. The technique is non-invasive and is effective under diverse environmental conditions. Recently, imaging fluorometers have been introduced that reveal variability in photosynthesis over the surface of a leaf or between individual plants. Most imaging instruments depend on continuous light or low frequency modulated light for fluorescence excitation, which imposes serious limitations on measurements of the fluorescence parameters, especially the minimum fluorescence (F₀) and variable fluorescence (F_V). Here, we describe a new instrument that combines the advantage of high frequency modulated light with two-dimensional imaging of chlorophyll fluorescence. The fluorometer produces dynamic images of chlorophyll fluorescence from leaves or plants, providing accurate mapping of F₀ and F_V, and non-photochemical quenching. A significant feature of the instrument is that it can record fluorescence images of leaves in daylight under field conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Assessing the poplar photochemical response to high zinc concentrations by image processing and statistical approach

Exposure of plants to high-heavy metals concentration inhibits multiple metabolic processes in plants and leads to an oxidative stress commonly referred as heavy metal ion toxicity. Chlorophyll a fluorescence has enhanced understanding of heavy metal ion action on the photosynthetic system. A rapid and non-invasive technique involving imaging of chlorophyll fluorescence is a useful tool for early detection of plant responses to heavy metal ion toxicity. In this work chlorophyll fluorescence emission and photochemical parameters in plants of Populus x euramericana clone I-214 were investigated by the portable Imaging PAM fluorometer at different days after soil treatment with zinc. Custom software for analysis of the photochemical parameters images has been developed in order to gain a better assessing of the plant performance in response of metal stress. The imaging analysis allowed visualizing heterogeneity in plant response to high zinc concentrations. The heterogeneity of images suggests spatial differences in photochemical activity and changes in the antenna down-regulation.     

Remote sensing of heterogeneity in photosynthetic efficiency, electron transport and dissipation of excess light in Populus deltoides stands under ambient and elevated CO2 concentrations, and in a tropical forest canopy, using a new laser-induced fluorescence transient device

Determining the spatial and temporal diversity of photosynthetic processes in forest canopies presents a challenge to the evaluation of biological feedbacks needed for improvement of carbon and climate models. Limited access with portable instrumentation, especially in the outer canopy, makes remote sensing of these processes a priority in experimental ecosystem and climate change research. Here, we describe the application of a new, active, chlorophyll fluorescence measurement system for remote sensing of light use efficiency, based on analysis of laser‐induced fluorescence transients (LIFT). We used mature stands of Populus grown at ambient (380 ppm) and elevated CO₂ (1220 ppm) in the enclosed agriforests of the Biosphere 2 Laboratory (B2L) to compare parameters of photosynthetic efficiency, photosynthetic electron transport, and dissipation of excess light measured by LIFT and by standard on‐the‐leaf saturating flash methods using a commercially available pulse‐modulated chlorophyll fluorescence instrument (Mini‐PAM). We also used LIFT to observe the diel courses of these parameters in leaves of two tropical forest dominants, Inga and Pterocarpus, growing in the enclosed model tropical forest of B2L. Midcanopy leaves of both trees showed the expected relationships among chlorophyll fluorescence‐derived photosynthetic parameters in response to sun exposure, but, unusually, both displayed an afternoon increase in nonphotochemical quenching in the shade, which was ascribed to reversible inhibition of photosynthesis at high leaf temperatures in the enclosed canopy. Inga generally showed higher rates of photosynthetic electron transport, but greater afternoon reduction in photosynthetic efficiency. The potential for estimation of the contribution of outer canopy photosynthesis to forest CO₂ assimilation, and assessment of its response to environmental stress using remote sensing devices such as LIFT, is briefly discussed.     

温室栽培对软枣猕猴桃叶片形态及叶绿素荧光特性的影响

【目的】深入研究温室栽培条件下软枣猕猴桃叶片的形态与叶绿素荧光特性,探明温室与露地栽培环境下叶片光形态建成的差异。【方法】以软枣猕猴桃品种‘佳绿’和‘魁绿’的5年生植株为试材,测定温室及露地栽培条件下不同叶龄叶片的叶绿素相对含量及叶绿素荧光参数,分析这些参数在温室与露地栽培条件下的差异。【结果】温室与露地栽培软枣猕猴桃的光形态建成均处于1~40 d叶龄间,不同栽培条件下的同一品种荧光特性趋于一致;花期后,不同栽培条件下的同一品种荧光特性差异较大。温室栽培的软枣猕猴桃叶片叶面积较大,叶绿素b含量较高,趋于阴生叶特性,以吸收光能为基础的性能指数等显著低于露地栽培,对光能的吸收捕获能力较强,但热耗散较高,用于电子传递的能量低于露地栽培,叶绿素荧光参数表现出对环境的适应性变化。【结论】软枣猕猴桃叶片的光形态建成时间为展叶后1~40 d,即在花期之前完成,不同栽培环境无明显差异;温室栽培软枣猕猴桃叶片的光形态建成与露地栽培具显著差异,叶面积明显增大;温室栽培在一定程度上改变了叶片的叶绿素荧光特性,降低了光合性能。     

Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance

The development of chlorophyll (Chl) a fluorescence imaging systems has greatly increased the versatility of Chl a fluorometry as a non-invasive technique for the investigation of photosynthesis in plants and algae. For example, systems that image at the microscopic level have made it possible to measure PSII photochemical efficiencies from chloroplasts within intact leaves and from individual algal cells within mixed populations, while systems that image over much larger areas have been used to investigate heterogeneous patterns of photosynthetic performance across leaves and in screening programmes that image tens or even hundreds of plants simultaneously. In addition, it is now practical to use fluorescence imaging systems as real-time, multi-channel fluorometers, which can be used to record continuous fluorescence traces from multiple leaves, plants, or algal cells. This paper discusses some of the theoretical and practical issues associated with the imaging of Chl a fluorescence and with Chl a fluorometry in general. This discussion includes a review of the most commonly used Chl a fluorescence parameters.