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Plants and algae have a love/hate relationship with light. As oxygenic photoautotrophic organisms, they require light for life; however, too much light can lead to increased production of damaging reactive oxygen species as byproducts of photosynthesis. In extreme cases, photooxidative damage can cause pigment bleaching and death, a phenomenon all too familiar to anyone who has tried to move a houseplant outdoors into full sunlight. The quantity of the light in natural environments can vary over several orders of magnitude and on a time scale that ranges from seconds to seasons. Because light is such an important environmental parameter, plants have evolved numerous biochemical and developmental responses to light that help to optimize photosynthesis and growth. For example, plants rely on photoreceptors such as phytochrome for shade avoidance responses. Some plants are able to adjust their capacity for harvesting sunlight through leaf and chloroplast movements. During long-term acclimation to changes in light intensity many algae and plants regulate the size of their light-harvesting pigment antennae through changes in gene expression and/or proteolysis. Large antennae are necessary for efficient light capture in limiting light, but they can be a liability when light is abundant or excessive. On a daily as well as seasonal basis most plants receive more sunlight than they can actually use for photosynthesis. Under these circumstances, regulation of light harvesting is necessary to balance the absorption and utilization of light energy, thereby minimizing the potential for photooxidative damage. Besides adjusting light absorption, algae and plants have ways of getting rid of excess light energy that has already been absorbed. This update will focus on protective non-photochemical mechanisms that quench singlet-excited chlorophylls (Chl) and harmlessly dissipate excess excitation energy as heat. These non-photochemical quenching (NPQ) processes occur in almost all photosynthetic eukaryotes, and they help to regulate and protect photosynthesis in environments in which light energy absorption exceeds the capacity for light utilization. We will summarize progress in understanding NPQ that has been made since the last Updatearticle on NPQ appeared in this journal (Horton et al., 1994). Possible fates of excited Chl. When Chl absorbs light it is excited from its ground state to its singlet excited state,1Chl*. From there it has several ways to relax back to the ground state. It can relax by emitting light, seen as fluorescence (1). Its excitation can be used to fuel photosynthetic reactions (2), or it can de-excite by dissipating heat (3); both of these mechanisms reduce the amount of fluorescence. They are therefore referred to as qP and NPQ of Chl fluorescence. Last,1Chl* can, by intersystem crossing, produce3Chl* (4), which in turn is able to produce1O2*, a very reactive oxygen species. Summary of Chl fluorescence quenching processes Summary of Chl fluorescence quenching processes Chl fluorescence measurement from an Arabidopsis leaf. In the presence of only weak measuring light the minimal fluorescence (F o) is seen. When a saturating light pulse is given, the photosynthetic light reactions are saturated and fluorescence reaches a maximum level (F m′). Upon continuous illumination with moderately excess light (750 μmol photons m−2sec1; growth light was 130 μmol photons m−2 sec−1), a combination of qP and NPQ lowers the fluorescence yield. NPQ (qE + qT + qI) can be seen as the difference between F m and the measured maximal fluorescence after a saturating light pulse during illumination (F m′). After switching off the light, recovery of F m′ within a few minutes reflects relaxation of the qE component of NPQ. NPQ can be divided into at least three different components according to their relaxation kinetics in darkness following a period of illumination, as well as their response to different inhibitors (Fig. 2; Horton and Hague, 1988). The major and most rapid component in most algae and plants is the pH- or energy-dependent component, qE. A second component, qT, relaxes within minutes and is more important in algae, but rather negligible in most plants during exposure to excess light. This component is due to the phenomenon of state transition, the uncoupling of LHCs from PSII. qT will not be considered further here because it does not seem to be important for photoprotection (Niyogi, 1999). The third component of NPQ shows the slowest relaxation and is the least defined. It is related to photoinhibition of photosynthesis and is therefore called qI. Absorption of sunlight that exceeds a plant's capacity for CO2 fixation results in a buildup of the thylakoid ΔpH that is generated by photosynthetic electron transport. The decrease in pH within the thylakoid lumen is an immediate signal of excessive light that triggers the feedback regulation of light harvesting by qE. The control by lumen pH allows induction or reversal of qE within seconds of a change in light intensity (see Fig. 2), which is fast enough to cope with natural fluctuations in light intensity that are due to, for example, passing clouds on a partly sunny day. The requirement for low lumen pH is evidenced by the inhibition of qE by uncouplers of the ΔpH such as nigericin. Screening for mutants with lower qE levels has uncovered several mutants that are defective in generation of the ΔpH due to defects in photosynthetic electron transport. In these mutants qP is also affected (Shikanai et al., 1999). However, low lumen pH that induces qE does not have to be generated by light-dependent reactions. Using isolated thylakoids it is possible to induce qE in darkness by simply lowering the pH of the buffer or by generating a ΔpH via ATP hydrolysis and reverse proton pumping by the ATP synthase (Gilmore and Yamamoto, 1992;Krieger et al., 1992). Intensive research during the past several years has led to a concept of the role of the ΔpH in qE. A decrease in lumen pH induces qE through protonation of PSII proteins and activation of xanthophyll synthesis via a xanthophyll cycle. Together, binding of protons and xanthophylls to specific sites in the PSII antenna causes a conformational change that switches a PSII unit into a quenched state with a short 1Chl* lifetime and a low fluorescence yield (Gilmore, 1997). In the following sections we will describe further the mechanistic details of this pH-dependent switch and the physiological significance of qE. Xanthophyll cycles. A, The violaxanthin cycle consists of the de-epoxidation of violaxanthin in high light to first antheraxanthin and then zeaxanthin, catalyzed by VDE; ZE catalyzes the reverse reaction. B, The diadinoxanthin cycle consists of the conversion of diadinoxanthin to diatoxanthin by diadinoxanthin de-epoxidase in high light and the reverse reaction in low light. C, In the recently discovered lutein-5,6-epoxide cycle, conversion of lutein-5, 6-epoxide to lutein might also be catalyzed by VDE. In plants the de-epoxidation reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE is a 43-kD nucleus-encoded protein that is localized in the thylakoid lumen (Bugos and Yamamoto, 1996). The purified VDE enzyme is activated by low pH (Eskling et al., 1997), and cloning of the VDE gene revealed that the enzyme has a Glu-rich region that may be protonated at low pH (Bugos and Yamamoto, 1996). Upon acidification of the lumen, VDE associates with the thylakoid membrane (Hager and Holocher, 1994) where it can interact with its substrate violaxanthin. VDE uses ascorbic acid (vitamin C) to reduce the epoxide group, and it has aK m for ascorbic acid that is strongly dependent on pH, probably because ascorbic acid rather than ascorbate is the actual cosubstrate (Bratt et al., 1995). A different enzyme, zeaxanthin epoxidase (ZE), catalyzes the epoxidation reactions that complete the violaxanthin cycle. ZE is a flavin adenine dinucleotide-containing, O2-dependent mono-oxygenase that uses reduced ferredoxin to epoxidize first zeaxanthin and then antheraxanthin (Bouvier et al., 1996). Because of its pH optimum of 8, ZE is thought to be located on the stromal side of the thylakoid membrane and to be constitutively active. The level of zeaxanthin is therefore determined by the activity of VDE compared with ZE, with rapid accumulation of zeaxanthin occurring upon activation of VDE in excessive light. ZE and VDE are the first known plant members of the lipocalin family, a diverse group of proteins that bind small lipophilic molecules and share a conserved tertiary structure of eight β-strands in a barrel configuration (Bugos et al., 1998). The amount of zeaxanthin synthesized via the violaxanthin cycle is highly correlated with the level of qE in a large number of plants under a variety of conditions (Demmig-Adams, 1990). In a similar manner, a correlation has been shown between qE and the conversion of diadinoxanthin to diatoxanthin in diatoms (Arsalane et al., 1994). Isolated thylakoids that are devoid of zeaxanthin have been observed to exhibit high levels of qE (Rees et al., 1992), but only at lumen pH values that are lower than those normally occurring in vivo. The requirement for the xanthophylls in qE has been investigated in vivo by using inhibitors and mutants. Dithiothreitol has been used extensively as a remarkably specific inhibitor of VDE and diadinoxanthin de-epoxidase. Blocking zeaxanthin synthesis in leaves with dithiothreitol results in inhibition of qE, the extent of inhibition depending on the plant species examined (Horton et al., 1994). Mutants that are unable to convert violaxanthin to antheraxanthin and zeaxanthin have been isolated from Arabidopsis andChlamydomonas (for review, see Niyogi, 1999). As in the inhibitor studies, lower levels of qE accompanied the lack of zeaxanthin in these npq1 mutants. Recent studies using antisense VDE in tobacco have confirmed the results obtained from mutant analyses (Chang et al., 2000). The alga Mantoniella squamata has an incomplete xanthophyll cycle, only leading to antheraxanthin in vivo, but still exhibits qE (Goss et al., 1998). This implies that the role of zeaxanthin in qE can be replaced by antheraxanthin in this alga. The involvement of antheraxanthin in plants had been proposed in earlier studies where zeaxanthin-independent qE could be explained by taking the amounts of antheraxanthin into account (Gilmore and Yamamoto, 1993). Therefore, it has become a common practice to calculate the level of de-epoxidation of a given organism as the amount of antheraxanthin and zeaxanthin in comparison with the total amount of antheraxanthin, zeaxanthin, and violaxanthin. In addition to antheraxanthin and zeaxanthin, a third xanthophyll, lutein, has also been implicated in qE. These suggestions were supported by studies on the Chlamydomonas lor1mutant, which only lacks lutein and loroxanthin, but shows lower qE than the wild type (Niyogi et al., 1997). A similar mutant in Arabidopsis, lut2, which is defective in the lycopene ε-cyclase and therefore lacks lutein, also has less qE (Pogson et al., 1998). Double mutants of Chlamydomonas or Arabidopsis that lack lutein and zeaxanthin are totally devoid of any qE and are very sensitive to high light (Niyogi et al., 1997, 2001). Furthermore, plants that overexpress the lycopene ε-cyclase have an increased lutein content and show a slight, but significant increase in the rate of qE induction, even though their xanthophyll cycle pool size is reduced in comparison with the wild type (Pogson and Rissler, 2000). It is interesting that a third kind of xanthophyll cycle involving lutein-5,6-epoxide has been found in a parasitic plant, Cuscuta reflexa (Fig. 3). In this plant neoxanthin is missing and is replaced by lutein-5, 6-epoxide. In high light, lutein-5,6-epoxide is de-epoxidized to lutein, presumably by VDE, which was previously shown to use lutein-5,6-epoxide as substrate (Bungard et al., 1999). The existence of this new cycle is consistent with the idea that lutein has a photoprotective function that the epoxide lacks. Although zeaxanthin is generally necessary for maximal qE in vivo, it is not sufficient. In mutants that accumulate zeaxanthin constitutively, qE must still be induced by a low pH (Niyogi, 1999). This demonstrates that the low pH has an additional role in qE, besides activation of the xanthophyll cycle. Lowering the pH in the thylakoid lumen not only activates the de-epoxidation of violaxanthin to zeaxanthin, but it is also necessary for a conformational change in the thylakoid membrane that can be monitored by absorbance changes. Two high light-induced absorbance changes in leaves or isolated thylakoids are associated with qE. One absorbance change occurs at 505 nm and is due to the conversion of violaxanthin to zeaxanthin. The second one at 535 nm (ΔA535) depends on both zeaxanthin and low pH and is thought to be due to a conformational change in the thylakoid membrane (Krause, 1973; Bilger and Björkman, 1994). qE is always accompanied by the ΔA535. Conformational changes have also been inferred from measurements of Chl fluorescence lifetime distributions, which depend on the molecular environment of the excited Chl (Gilmore, 1997). In these experiments the presence of a ΔpH alone causes a lifetime shift from approximately 2 to 1.6 ns. This shift likely reflects a protonation-dependent conformational change that is independent of zeaxanthin. When both ΔpH and zeaxanthin are present, a fluorescence lifetime component at 0.4 ns appears at the expense of the 1.6 ns component. The amount of the 0.4 ns component is proportional to qE. Together, the absorbance and Chl fluorescence lifetime results suggest that a conformational change due to binding of protons and xanthophylls (maybe zeaxanthin) is necessary for qE. Several LHC proteins associated with PSII have been implicated in qE. In particular the minor LHC proteins CP29 and CP26 were suggested to be involved in qE based on the relative enrichment of associated xanthophyll cycle pigments (Bassi et al., 1997) and binding ofN,N′-dicyclohexylcarbodiimide (Walters et al., 1994), an inhibitor of qE that reacts with proton active residues. However, these proteins have not been found in some organisms that exhibit qE such as Mantoniella and diatoms. To identify proteins involved in qE Arabidopsis mutants have been isolated that are defective in qE, but have normal xanthophyll levels (Li et al., 2000; Peterson and Havir, 2000). Characterization of one of these mutants, npq4-1, revealed that a PSII protein, PsbS, is essential for qE (Li et al., 2000). PsbS belongs to the LHC protein superfamily, but it has four transmembrane helices instead of three and different pigment-binding characteristics (Funk et al., 1995). Despite the absence of the PsbS protein, light harvesting is not impaired in the npq4-1 mutant and the levels of the other LHC proteins are normal. However, in addition to lacking qE, npq4-1 also lacks the conformational change monitored by ΔA535 (Li et al., 2000). The pH- and xanthophyll-dependent conformational change and the PsbS protein are necessary for qE, but the actual biophysical mechanism of 1Chl* de-excitation is still unknown. The central and long-standing question is whether the involvement of xanthophylls is direct or indirect. The xanthophylls may act indirectly as allosteric regulators of the LHCs that cause a switch from light harvesting to energy dissipation (qE; Horton et al., 2000). In this case the conformational change must somehow facilitate1Chl* de-excitation, which may occur by internal conversion of Chl itself to the ground state, releasing excitation energy as heat. Isolated, detergent-solubilized LHCs can exhibit pH-dependent Chl fluorescence quenching, which is inhibited by violaxanthin and promoted by zeaxanthin (Ruban et al., 1997). On the other hand, xanthophylls may directly de-excite1Chl*. This is theoretically possible according to recent spectroscopic experiments, which showed that isolated xanthophyll cycle pigments possess a lowest singlet excited state that is below that of 1Chl* (Polı́vka et al., 1999; Frank et al., 2000). However, because both violaxanthin and zeaxanthin could potentially accept energy from1Chl*, these results do not explain why the xanthophyll cycle is necessary for qE. In vivo, other factors such as different binding sites for zeaxanthin and violaxanthin, as well as distance and orientation of these xanthophylls relative to Chl may be important in energy Therefore, rather than between zeaxanthin and violaxanthin are likely to be For example, the xanthophylls that are important for qE antheraxanthin, lutein, and all have a de-epoxidized (Fig. in to violaxanthin, and other A for qE. A, In limiting light or darkness quenching B, In high light a likely several bind to PsbS and LHC a shift in Chl fluorescence lifetime to 1.6 ns. C, A quenching with a different as is when zeaxanthin and protons are the Chl fluorescence lifetime to 0.4 ns. of violaxanthin to zeaxanthin occurs more than might bind to the as violaxanthin or a different When the light has the PSII proteins are the epoxidation of zeaxanthin to violaxanthin is In the of from A is not possible in vivo, but can be induced by the pH in the thylakoid The is based in on by Horton (Horton et al., The mutants have been for the photoprotective function of qE during high light The Arabidopsis mutants npq1 and are more sensitive to photoinhibition than the wild type in a scale of et al., and Niyogi, that qE normally to protect PSII. The photoprotective of qE may be due to production of and other reactive oxygen species. qE may also the of the electron and the of the lumen, which are known to PSII to After several in high light, the npq1 mutant showed more photooxidative bleaching and and Niyogi, 1999). to antisense tobacco plants that lack VDE showed a significant increase in photoinhibition and a decrease in pigment content when to high light or a combination of light and in a growth et al., 2001). When npq1 lut2, an Arabidopsis mutant missing zeaxanthin and lutein, into high light even more photooxidative bleaching and was (Niyogi et al., 2001). These results that xanthophylls have a function not only in qE, but also in the of the thylakoid membrane photooxidative damage. may directly protect the thylakoid membrane are in that are reactions. within leaves with the in leaves of but not in that the photoprotective role of zeaxanthin is not to the LHCs et al., 2000). may be an important in the thylakoid membrane where it could reactive oxygen species and/or reactions. and lutein the in made from in response to a et al., 1999). the of zeaxanthin and lutein has important not only for but also for of the where these xanthophylls are found could also have a function in the This xanthophyll has been shown to decrease the of the membrane and 1997), and a decrease in could be important by lowering the of reactive oxygen species the In qE PSII high light and fluctuations in light xanthophylls have an additional photoprotective role in high light. Although the light of the mutants of Arabidopsis and the VDE antisense plants of tobacco has been these mutants are remarkably of light. In leaves of the mutants are to high light or et al., 2000; et al., that other important photoprotective mechanisms or other can at least for the lack of qE and/or Under more light qE is replaced by a component of called qI. In to qE, is much less and might be due to a of photoprotection and Chl fluorescence measurements can help to between photoprotective mechanisms and The fluorescence level in the (see Fig. 2), is in direct to the maximal F by the photoprotective quenching qE, photoinhibition normally the F level the F m level (Gilmore et al., 1996). plants show an acclimation to the by the xanthophyll pool as well as by an increased of zeaxanthin and antheraxanthin that is associated with et al., 1999). to that dissipate excess excitation energy (Gilmore and 2000). of Chl fluorescence lifetime of this state have revealed lifetime changes that are similar to those observed during qE, but that are only at (Gilmore and 2000). and reversal of in these leaves may major of in the thylakoid Some of the induced by low is actually as qE because it is an this kind of can be (Gilmore and Björkman, 1995). It may be due to the of the ΔpH by the reverse proton pumping catalyzed by the (Gilmore, 1997). Therefore, in to normal qE it relaxes only in has since the last on this was in this journal (Horton et al., 1994). on physiological studies, the function for qE to be the of PSII from However, when qE is other mechanisms are able to for long-term at least in the absence of additional studies using qE mutants are necessary to the of qE and to other important photoprotective mechanisms that qE. The PsbS protein is an essential component of the mechanism of qE. PsbS itself may be the of qE in the or it may function in with other LHC To PsbS actually in qE, its within the as well as possible and pigment-binding sites within PsbS have to be Characterization of additional mutants or the use of reverse may into the involvement of other new in the has been the that both zeaxanthin and violaxanthin are potential of excitation energy from In the it will be important to experiments that measurement of the energy levels of xanthophylls in their protein environment and to there is a direct energy from Chl to a The of new and diverse from to to will be necessary to qE, a response of photosynthetic to excess light We and for results to and and for of the We to we were not able to due to

Reproducibility is a current concern for everyone involved in the conduct and publication of biomedical research. Recent attempts testing reproducibility, particularly the reproducibility project in cancer biology published in elife (https://elifesciences.org/collections/9b1e83d1/reproducibility-project-cancer-biology), have exposed major difficulties in repeating published preclinical experimental work. It is thought that some of these difficulties relate to uncertainty about the provenance of tools, lack of clarity in methodology and use of inappropriate approaches for analysis; the latter particularly related to untoward manipulation of images. In the past, some of these so-called untoward practices were considered the ‘norm’; however, today, the landscape is different. The expectations, not only of the readers of the published scientific word but also of the publishers and funders of research, have changed. This collective group now expects that any published data should be reproducible; but for this to be possible, experimental detail, confirmation of selectivity and quality of reagents/tools, analytical and statistical methods used need to be described adequately. Two powerful methodologies often used to support researchers' findings allow the detection of changes in protein expression, that is, immunoblotting (widely known as Western blotting) and immunohistochemistry. Undeniably, as a result of unintentional mistakes (often related to lack of antibody specificity; Baker, 2015), but, in some cases, deliberate alterations and questionable interpretations of results, the use of these two methods has led to many high profile retractions. Indeed, such images have driven the retractions that have occurred in BJP over the last two years. Today, immunoblotting and immunohistochemistry serve as primary methodologies for the detection and quantification of molecular signalling pathways and identification of therapeutic targets. This necessitates clear guidance for the application of these techniques, the need for controls (both positive and negative) and the most appropriate methods for quantification. Indeed, this need has spawned a number of initiatives to support researchers in assessing the validity of antibody resources including antibodypedia (Bjorling and Uhlen, 2008) and the resources available within ‘The Human Protein Atlas’ (Thul et al., 2017). The aim of this article is to outline the rationale for, and the expectations of, the BJP with respect to work published in the Journal that includes immunoblotting or immunohistochemical data. In creating these guidelines, our aim is to reduce potential misinterpretations and to maximise the communication and transparency of essential information, particularly with respect to the methodologies employed. We have generated the guidelines below for the benefit of authors, editors and reviewers. While we recognise other recently published guidelines (Uhlen et al., 2016) and indeed we have incorporated some of the advice provided in such reports, we focus, here, on the evidence required for publication in BJP. These guidelines join a series published in BJP regarding the reporting of animal experiments through adoption of the ARRIVE guidelines (McGrath et al., 2015; McGrath and Lilley, 2015), experimental design and analysis (Curtis et al., 2015) and data sharing and presentation (George et al., 2017) in preclinical pharmacology. We would be delighted if other journals were to also use these guidelines. If an antibody has been characterised previously, a citation must be included. This characterization should fulfil the requirements detailed in the recent guidelines generated by ‘The International Working Group for Antibody Validation’ and should take the form of at least, but preferably more than, one of the ‘five conceptual pillars of antibody validation’ described in the guidelines (Uhlen et al., 2016). We also ask authors to check currently available databases for known issues with selectivity. Currently, we highly recommend the NIH-established Research Resource Identification Protocol (RRID) at https://scicrunch.org/resources/Cell%20Lines/search, which provides a unique identifier for the antibody that enables reproducibility studies through clear indication of provenance. Although encouraged, the inclusion of an RRID is not currently mandatory for BJP; we envisage that a similar level of individualization will become essential for publication in the near future. To support the validity of conclusions emanating from immunoblotting or immunohistochemical observations (as with the majority of biological assays), it is important to conduct both positive and negative controls – although for some proteins, certain positive and negative controls are unknown or such materials are not available, thereby precluding such assessments. However, there are a number of other ‘method’ controls that are always possible and which should be included in assay protocols. We recognise that there may be no perfect control that provides confidence beyond doubt (Torlakovic et al., 2015). In this digital age, a growing concern amongst the scientific community is that images for publication are not being accurately presented, that is, the problem of unintentional and/or inappropriate manipulation of images (Cromey, 2010). For example, while it is usually acceptable to crop an image to simplify the information, choosing to crop out oversaturated regions and/or areas/bands displaying non-specific immunoreactivity is not acceptable. BJP requires submission of full immunoblot scans and immunohistochemical/fluorescent images, from which figures have been generated. These scans/images should be included as an additional file for the review process and will be used by the Editors and reviewers when assessing the manuscript, but need not be published. In instances where uncertainty occurs regarding image compilation and assembly during the review process, the BJP Editorial Office makes use of freely available Office of Research Integrity Forensic tools (https://ori.hhs.gov/forensic-tools), developed by the US Department of Health and Human Services. Conforming to these stipulations will ensure that reviewers and readers can confirm that the band identified is at the correct molecular size (or allow some interpretation of post-translational modifications, for example) and to determine the selectivity of the antibody used. For any antibody (including secondary antibodies) used, the Methods section should include the following:

Early identification of high-risk disease could greatly reduce both mortality and morbidity due to oral cancer. We describe a simple handheld device that facilitates the direct visualization of oral-cavity fluorescence for the detection of high-risk precancerous and early cancerous lesions. Blue excitation light (400 to 460 nm) is employed to excite green-red fluorescence from fluorophores in the oral tissues. Tissue fluorescence is viewed directly along an optical axis collinear with the axis of excitation to reduce inter- and intraoperator variability. This robust, field-of-view device enables the direct visualization of fluorescence in the context of surrounding normal tissue. Results from a pilot study of 44 patients are presented. Using histology as the gold standard, the device achieves a sensitivity of 98% and specificity of 100% when discriminating normal mucosa from severe dysplasia/carcinoma in situ (CIS) or invasive carcinoma. We envisage this device as a suitable adjunct for oral cancer screening, biopsy guidance, and margin delineation.

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, light-harvesting 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.

Abstract We have extended a conventional photosynthesis model to simulate field and laboratory measurements of chlorophyll fluorescence at the leaf scale. The fluorescence paramaterization is based on a close nonlinear relationship between the relative light saturation of photosynthesis and nonradiative energy dissipation in plants of different species. This relationship diverged only among examined data sets under stressed (strongly light saturated) conditions, possibly caused by differences in xanthophyll pigment concentrations. The relationship was quantified after analyzing data sets of pulse amplitude modulated measurements of chlorophyll fluorescence and gas exchange of leaves of different species exposed to different levels of light, CO 2 , temperature, nitrogen fertilization treatments, and drought. We used this relationship in a photosynthesis model. The coupled model enabled us to quantify the relationships between steady state chlorophyll fluorescence yield, electron transport rate, and photosynthesis in leaves under different environmental conditions.

There is extensive interest in automating the collection, organization and analysis of biological data. Data in the form of images present special challenges for such efforts. Since fluorescence microscope images are a primary source of information about the location of proteins within cells, we have set as a long-term goal the building of a knowledge base system that can interpret such images in online journals. To this end, we first developed a robot that searches online journals and finds fluorescence microscope images of individual cells. We then characterized the applicability of pattern classification methods we have previously used on images obtained under controlled conditions to images from different sources and to images subjected to manipulations commonly performed during publication. The results indicate the feasibility of developing search engines to find fluorescence microscope images depicting particular subcellular patterns.

In the last century the production and application of halides assumed an ever greater importance. In the fields of medicine, dentistry, plastics, pesticides, food, photography etc many new halogen containing compounds have come into everyday use. In an analogous manner many techniques for the detection and determination of halogen compounds and ions have been developed with scientific journals reporting ever more sensitive methods. The 19th century saw the discovery of what is now thought of as a classical method for halide determination, namely the quenching of fluorescence. However, little analytically was done until over 100 years after its discovery, when the first halide sensors based on the quenching of fluorescence started to emerge. Due to the typical complexity of fluorescence quenching kinetics of optical halide sensors and their lack of selectivity, they have found little if any place commercially, despite their sensitivity, where other techniques such as ion-selective electrodes, x-ray fluorescence spectroscopy and colorimetry have dominated the analytical market. In this review article the author summarizes the relevant theory and work to date for halide sensing using fluorescence quenching methods and outlines the future potential that fluorescence quenching based optical sensors have to offer in halide determination.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g. , biomedicine, biophysics, and chemistry. By time-correlation of the fluorescence fluctuations induced by molecules diffusing through a focused light, FCS can quantitatively evaluate the concentration, diffusion coefficient, and interaction of the molecules in vitro or in vivo . In this review, the basic principle and implementation of FCS are introduced. Then, the advances of FCS variants are reviewed, covering dual-color FCCS, multi-focus FCS, pair correlation function (pCF), scanning FCS, focus-reduced FCS, SPIM-FCS, and inverse-FCS. Besides, the applications of FCS are demonstrated with the measurement of local concentration, hydrodynamic radius, diffusion coefficient, and the interaction of different molecules. Lastly, a discussion is given by summarizing the pros and cons of different FCS techniques, as well as the outlooks and perspectives of FCS.

The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.

BACKGROUND AND AIMS: Salinity is a widespread soil problem limiting productivity of cereal crops worldwide. Rice is particularly sensitive to salt stress during the seedling stage, with consequent poor crop establishment, as well as during reproduction where salinity can severely disrupt grain formation and yield. Tolerance at the seedling stage is weakly associated with tolerance during reproduction. Physiological responses to salinity were evaluated for contrasting genotypes, during the seedling and reproductive stages. METHODS: Three rice genotypes differing in their tolerance of salinity were evaluated in a set of greenhouse experiments under salt stress during both seedling stage and reproduction. KEY RESULTS: Photosynthetic CO2 fixation, stomatal conductance (gs) and transpiration decreased substantially because of salt stress, but with greater reduction in the sensitive cultivar IR29. The tolerant lines IR651 and IR632 had more responsive stomata that tended to close faster during the first few hours of stress, followed by partial recovery after a brief period of acclimation. However, in the sensitive line, gs continued to decrease for longer duration and with no recovery afterward. Chlorophyll fluorescence measurements revealed that non-photochemical quenching increased, whereas the electron transport rate decreased under salt stress. Salt-tolerant cultivars exhibited much lower lipid peroxidation, maintained elevated levels of reduced ascorbic acid and showed increased activities of the enzymes involved in the reactive oxygen scavenging system during both developmental stages. CONCLUSIONS: Upregulation of the anti-oxidant system appears to play a role in salt tolerance of rice, with tolerant genotypes also maintaining relatively higher photosynthetic function; during both the vegetative and reproductive stages.

This report summarizes the work performed during the past two years at the National Institute of Standards and Technology (NIST) in the refinement and formal definition of the MESF unit of fluorescence intensity. In addition to the theory underlying the MESF unit, considerations of error analysis are also presented. The details of this work may be found in the three publications of the NIST Journal of Research (www.nist.gov) listed as the references 2-4. The use of the fluorescence intensity unit provides a tool to compare quantitative fluorescence intensity measurements over time and across platforms.

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Förster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

In recent years, solar-induced chlorophyll fluorescence (SIF) retrieved from space borne spectrometers has been extensively used as a proxy for terrestrial photosynthesis at relatively sparse temporal and spatial scales. The near-infrared band of the recently launched TROPOspheric Monitoring Instrument (TROPOMI) features the required spectral resolution and signal-to-noise ratio to retrieve SIF in a spectral range devoid of atmospheric absorption features. We find that initial TROPOMI spectra meet high expectations for a substantially improved spatio-temporal resolution (up to 7 km × 3.5 km pixels with daily revisit), representing a step change in SIF remote sensing capabilities. However, interpretation requires caution, as the broad range of viewing-illumination geometries covered by TROPOMI's 2600 km wide swath needs to be taken into account. A first inter-sensor comparison with OCO-2 (Orbiting Carbon Observatory-2) SIF shows excellent agreement, underscoring the high quality of TROPOMI's SIF retrievals and the notable radiometric performance of the instrument. PLAIN LANGUAGE SUMMARY: Photosynthesis is the most essential process for life on Earth, but gradually changing environmental conditions such as increasing concentrations of atmospheric trace gases, rising temperatures or reduced water availability could adversely affect the photosynthetic productivity. The recently launched TROPOspheric Monitoring Instrument (TROPOMI) is designed to monitor atmospheric trace gases and air pollutants with an unprecedented resolution in space and time, while its radiometric performance also permits us to see a weak electromagnetic signal emitted by photosynthetically active vegetation - solar induced chlorophyll fluorescence (SIF). Mounting evidence suggests that SIF observations from satellite instruments augment our abilities to track the photosynthetic performance and carbon uptake of terrestrial vegetation. In this study, we present the first TROPOMI SIF retrievals, largely outperforming previous and existing capabilities for a spatial continuous monitoring of SIF from space.

Chemical tools that allow the real-time monitoring of organ function and the visualisation of organ-related processes at the cellular level are of great importance in biological research. The upregulation/downregulation of specific biomarkers is often associated with the development of organ related diseases. Small-molecule fluorescent probes have the potential to create advances in our understanding of these disorders. Viable probes should be endowed with a number of key features that include high biomarker sensitivity, low limit of detection, fast response times and appropriate in vitro and in vivo biocompatibility. In this tutorial review, we discuss the development of probes that allow the targeting of organ related processes in vitro and in vivo. We highlight the design strategy that underlies the preparation of various promising probes, their optical response to key biomarkers, and proof-of-concept biological studies. The inherent drawbacks and limitations are discussed as are the current challenges and opportunities in the field. The hope is that this tutorial review will inspire the further development of small-molecule fluorescent probes that could aid the study of pathogenic conditions that contribute to organ-related diseases.

Chlorophyll a fluorescence (ChlF) has been used for decades to study the organization, functioning, and physiology of photosynthesis at the leaf and subcellular levels. ChlF is now measurable from remote sensing platforms. This provides a new optical means to track photosynthesis and gross primary productivity of terrestrial ecosystems. Importantly, the spatiotemporal and methodological context of the new applications is dramatically different compared with most of the available ChlF literature, which raises a number of important considerations. Although we have a good mechanistic understanding of the processes that control the ChlF signal over the short term, the seasonal link between ChlF and photosynthesis remains obscure. Additionally, while the current understanding of in vivo ChlF is based on pulse amplitude-modulated (PAM) measurements, remote sensing applications are based on the measurement of the passive solar-induced chlorophyll fluorescence (SIF), which entails important differences and new challenges that remain to be solved. In this review we introduce and revisit the physical, physiological, and methodological factors that control the leaf-level ChlF signal in the context of the new remote sensing applications. Specifically, we present the basis of photosynthetic acclimation and its optical signals, we introduce the physical and physiological basis of ChlF from the molecular to the leaf level and beyond, and we introduce and compare PAM and SIF methodology. Finally, we evaluate and identify the challenges that still remain to be answered in order to consolidate our mechanistic understanding of the remotely sensed SIF signal.

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.

SIGNIFICANCE: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. AIM: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. APPROACH: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. CONCLUSIONS: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

Scanning confocal microscopes offer improved rejection of out-of-focus noise and greater resolution than conventional imaging. In such a microscope, the imaging and condenser lenses are identical and confocal. These two lenses are replaced by a single lens when epi-illumination is used, making confocal imaging particularly applicable to incident light microscopy. We describe the results we have obtained with a confocal system in which scanning is performed by moving the light beam, rather than the stage. This system is considerably faster than the scanned stage microscope and is easy to use. We have found that confocal imaging gives greatly enhanced images of biological structures viewed with epifluorescence. The improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections.

The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy.