Autofluorescence: Biological functions and technical applications (original) (raw)

Secondary Metabolite Localization by Autofluorescence in Living Plant Cells

Molecules, 2015

Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

Chlorophyll Fluorescence in Plant Biology

2012

Several molecules absorb light energy which they emit after a time difference (lifetime) as radiation energy. Molecules remain at a low energy level or the ground electronic singlet state (So) or the lowest vibrational level at room temperature (Noomnarm and Clegg, 2009). On absorption of a photon, the molecule is excited from So to the first electronic excited singlet state S1 within< 10-15 s-1 (Figure 1). These molecules can also be transferred to higher energy levels (S2 to Sn) also.

The use of chlorophyll fluorescence nomenclature in plant stress physiology

Photosynthesis Research, 1990

During recent years there has been remarkable progress in the understanding and practical use of chlorophyll fluorescence in plant science. This 'renaissance' of chlorophyll fluorescence was induced by the urgent need of applied research (like plant stress physiology, ecophysiology, phytopathology etc.) for quantitative, non-invasive, rapid methods to assess photosynthesis in intact leaves. Recent developments of suitable instrumentation and methodology have substantially increased these possibilities. Actually, a vast amount of knowledge on chlorophyll fluorescence had already accumulated over more than 50 years, since the discovery of the Kautsky effect in 1931 (Kautsky and Hirsch 1931) (for reviews, see e.g., Lavorel and Etienne 1977, Renger and Schreiber 1986). On the one hand this knowledge was mechanistic, resulting from biophysically oriented basic research. On the other hand it was phenomenological, originating from applied plant physiological research. Until recently the phenomenology of whole leaf chlorophyll fluorescence appeared far too complex to find serious attention of biophysicists. Thus, for a long time, there was a gap between applied and basic research in chlorophyll fluorescence. Developments in instrumentation (Ogren and Baker 1985, Schreiber 1986, Schreiber et al. 1986) and methodology , Krause et al. 1982, Quick and Horton 1984, Dietz et al. 1985, Demmig et al. 1987, Weis and Berry 1987, Genty et al. 1989) has succeeded in closing this gap and bringing these two disciplines into sufficiently close contact and in mutually stimulating interaction. Consequently the present "renaissance" of chlorophyll fluores-cence may be the product of a fruitful dynamic interaction between three different research disciplines, i.e., basic and applied research linked to new developments in instrumentation and methodology (see scheme in . As a result, measuring chlorophyll fluorescence has become a very attractive means of obtaining rapid, semiquantitative information on photosynthesis, used by an increasing number of researchers not only in the laboratory but also in the field. The wide range of possible applications is reflected by the broad spectrum of contributions to this issue of Photosynthesis Research.

Frequently asked questions about chlorophyll fluorescence, the sequel

Photosynthesis Research, 2016

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V /F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor Hazem M. Kalaji and Gert Schansker have contributed equally to this paper.

Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions

Acta Physiologiae Plantarum, 2016

Plants living under natural conditions are exposed to many adverse factors that interfere with the photosynthetic process, leading to declines in growth, development, and yield. The recent development of Chlorophyll a fluorescence (ChlF) represents a potentially valuable new approach to study the photochemical efficiency of leaves. Specifically, the analysis of fluorescence signals provides detailed information on the status and function of Photosystem II (PSII) reaction centers, lightharvesting antenna complexes, and both the donor and acceptor sides of PSII. Here, we review the results of fast ChlF analyses of photosynthetic responses to environmental stresses, and discuss the potential scientific and practical applications of this innovative methodology. The recent availability of portable devices has significantly expanded the potential utilization of ChlF techniques, especially for the purposes of crop phenotyping and monitoring.

In Vivo Measurements of Light Emission in Plants

Photosynthesis: Open Questions and What We Know Today, Allakhverdiev S. I., Rubin A. B., Shuvalov V.A. (eds.), 2014

There are several types of light emission in plants: prompt fluorescence, delayed fluorescence, thermoluminescence, and phosphorescence. This chapter focuses on two of them: prompt and delayed fluorescence. Chlorophyll a fluorescence measurements have been used for more than 80 years to study photosynthesis; since 1961, it has been used, particularly, for the analysis of PhotosystemII (PS II). Fluorescence is now used routinely in agricultural and biological research where many measured and calculated parameters are used as biomarkers or indicators of plant tolerance to different abiotic and biotic stress. This has been made possible by the rapid development of new fluorometers. Most of these instruments are mainly based on two different operational principles for the measurement of variable chlorophyll a fluorescence: (1) pulse-amplitude-modulated (PAM) excitation followed by measurement of prompt fluorescence and (2) a strong continuous actinic excitation leading to prompt fluorescence. In addition to fluorometers, other instruments have been developed to measure other signals, such as delayed fluorescence, originating mainly from PS II, and light-induced absorbance changes due to the photo-oxidation of the reaction center P700 of PS I, measured as absorption decrease (photobleaching) at about 705 nm, or increase at 820 nm. This chapter includes technical and theoretical basis of newly developed instruments that allow for simultaneous measurement of the prompt fluorescence (PF) and the delayed fluorescence (DF) as well as some other In vivo MEASUREMENTS OF LIGHT EMISSION IN PLANTS 3 parameters. Special emphasis is given here to a description of comparativemeasurements on PF and DF. Since DF is much less used and less known than PF, it is discussed in greater details; it has great potential to provide useful, and qualitatively new information on the back reactions of PS II electron transfer. This chapter, which also deals with the history of fluorometers, is dedicated to David Walker (1928–2012), who was a pioneer in the field of photosynthesis and chlorophyll fluorescence.

In Vivo Measurements of Light Emission in Plantsa

There are several types of light emission in plants: prompt fluorescence,delayed fluorescence, thermoluminescence, and phosphorescence. This chapter focuses on two of them: prompt and delayed fluorescence. Chlorophyll a fluorescence measurements have been used for more than 80 years to study photosynthesis; since 1961, it has been used, particularly, for the analysis of PhotosystemII (PS II). Fluorescence is now used routinely in agricultural and biological research where many measured and calculated parameters are used as biomarkers or indicators of plant tolerance to different abiotic and biotic stress. This has been made possible by the rapid development of new fluorometers. Most of these instruments are mainly based on two different operational principles for the measurement of variable chlorophyll a fluorescence: (1) pulse-amplitude-modulated (PAM) excitation followed by measurement of prompt fluorescence and (2) a strong continuous actinic excitation leading to prompt fluorescence. In addition to fluorometers, other instruments have been developed to measure other signals, such as delayed fluorescence, originating mainly from PS II, and light-induced absorbance changes due to the photo-oxidation of the reaction center P700 of PS I, measured as absorption decrease (photobleaching) at about 705 nm, or increase at 820 nm. This chapter includes technical and theoreticalbasis of newly developed instruments that allow for simultaneous measurement of the promptfluorescence (PF) and the delayed fluorescence (DF) as well as some otherparameters. Special emphasis is given here to a description of comparativemeasurements on PF and DF. Since DF is much less used and less known than PF, it is discussed in greater details; it has great potential to provide useful, and qualitatively new information on the back reactions of PS II electron transfer. This chapter, which also deals with the history of fluorometers, is dedicated to David Walker (1928–2012), who was a pioneer in the field of photosynthesis and chlorophyll fluorescence.

Experimental in vivo measurements of light emission in plants: a perspective dedicated to David Walker

Photosynthesis Research, 2012

This review is dedicated to David Walker , a pioneer in the field of photosynthesis and chlorophyll fluorescence. We begin this review by presenting the history of light emission studies, from the ancient times. Light emission from plants is of several kinds: prompt fluorescence (PF), delayed fluorescence (DF), thermoluminescence, and phosphorescence. In this article, we focus on PF and DF. Chlorophyll a fluorescence measurements have been used for more than 80 years to study photosynthesis, particularly photosystem II (PSII) since 1961. This technique has become a regular trusted probe in agricultural and biological research. Many measured and calculated parameters are good biomarkers or indicators of plant tolerance to different abiotic and biotic stressors. This would never have been possible without the rapid development of new fluorometers. To date, most of these instruments are based mainly on two different operational principles for measuring variable chlorophyll a fluorescence: (1) a PF signal produced following a pulseamplitude-modulated excitation and (2) a PF signal emitted during a strong continuous actinic excitation. In addition to fluorometers, other instruments have been developed to measure additional signals, such as DF, originating from PSII, and light-induced absorbance changes due to the photooxidation of P700, from PSI, measured as the absorption decrease (photobleaching) at about 705 nm, or

Demonstration of plant fluorescence by imaging technique and Intelligent FluoroSensor

Education and Training in Optics and Photonics: ETOP 2015, 2015

Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called 'Kautsky effect', is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.