The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live‐cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, (...) the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. (shrink)

What kind of symbiosis between archaeon and bacterium gave rise to their eventual merger at the origin of the eukaryotes? I hypothesize that conditions favouring bacterial uptake were based on exchange of intermediate carbohydrate metabolites required by recurring changes in availability and use of the two different terminal electron chain acceptors, the bacterial one being oxygen. Oxygen won, and definitive loss of the archaeal membrane potential allowed permanent establishment of the bacterial partner as the proto‐mitochondrion, further metabolic integration and highly (...) efficient ATP production. This represents initial symbiogenesis, when crucial eukaryotic traits arose in response to the archaeon‐bacterium merger. The attendant generation of internal reactive oxygen species (ROS) gave rise to a myriad of further eukaryotic adaptations, such as extreme mitochondrial genome reduction, nuclei, peroxisomes and meiotic sex. Eukaryotic origins could have started with shuffling intermediate metabolites as is still essential today. (shrink)

The repertoire of acceptor glycoproteins for concanavalin A expressed by a cultured tumour cell reflects the normal developmental lineage from which it was derived, as well as the degree of maturation along that lineage. Antibodies to this particular set of glycoproteins show a considerable specificity towards normal differentiation antigens which are often preferentially associated with the less mature intermediates of the corresponding pathway. In addition, comparisons between ‘immature’ and ‘mature’ tumour cells can be used to identify glycoproteins associated with (...) specific differentiated functions such as secretion. Thus tumour cell glycoproteins provide useful probes for analysis of the molecular biology of development and differentiation. (shrink)

Aspects of peroxisome evolution, uncoupling, carnitine shuttles, supercomplex formation, and missing neuronal fatty acid oxidation (FAO) are linked to reactive oxygen species (ROS) formation in respiratory chains. Oxidation of substrates with high FADH2/NADH (F/N) ratios (e.g., FAs) initiate ROS formation in Complex I due to insufficient availability of its electron acceptor (Q) and reverse electron transport from QH2, e.g., during FAO or glycerol‐3‐phosphate shuttle use. Here it is proposed that the Q‐cycle of Complex III contributes to enhanced ROS formation (...) going from low F/N ratio substrates (glucose) to high F/N substrates. This contribution is twofold: 1) Complex III uses Q as substrate, thus also competing with Complex I; 2) Complex III itself will produce more ROS under these conditions. I link this scenario to the universally observed Complex III dimerization. The Q‐cycle of Complex III thus again illustrates the tension between efficient ATP generation and endogenous ROS formation. This model can explain recent findings concerning succinate and ROS‐induced uncoupling. (shrink)

Controversies in food and agriculture abound, with many portrayed as conflicts between polarized viewpoints. Framing such controversies as dichotomies, however, can at times obscure what might be a plurality of views and potential common ground on the subject. We used Q methodology to explore stakeholders’ views about pesticide safety, agricultural worker exposure, and human health concerns in the tree fruit industry of central Washington State. Using a purposive sample of English and Spanish-speaking agricultural workers, industry representatives, state agencies, educators, and (...) advocates, participants sorted 45 statements on pesticide use and perceived human safety risks in the tree fruit industry in 2011. We used PQMethod 2.33 statistical software program to identify viewpoints, based on differences between how participants sorted the statements. The results revealed three distinct viewpoints among 38 sorters that explained 52 percent of the variance. The viewpoints included the: skeptics who expressed concern over the environmental and human health impacts of pesticide use; acceptors who acknowledged inherent risks for using pesticides but saw the risks as known, small and manageable; and incrementalists who prioritized opportunities to introduce human capital and technological improvements to increase agricultural worker safety. We then brought representatives with these different viewpoints together to analyze the results of the Q study, and to brainstorm mutually acceptable improvements to health and safety in tree fruit orchards. In describing and analyzing this case study, we argue that Q methodology can serve as one potentially effective tool for collaborative work, in this case facilitating a process of orchard safety improvements despite perceived stakeholder polarization. (shrink)

Inner membranes of mitochondria are extensively folded, forming cristae. The observed overall correlation between efficient eukaryotic ATP generation and the area of internal mitochondrial inner membranes both in unicellular organisms and metazoan tissues seems to explain why they evolved. However, the crucial use of molecular oxygen (O2) as final acceptor of the electron transport chain is still not sufficiently appreciated. O2 was an essential prerequisite for cristae development during early eukaryogenesis and could be the factor allowing cristae retention upon (...) loss of mitochondrial ATP generation. Here I analyze illuminating bacterial and unicellular eukaryotic examples. I also discuss formative influences of intracellular O2 consumption on the evolution of the last eukaryotic common ancestor (LECA). These considerations bring about an explanation for the many genes coming from other organisms than the archaeon and bacterium merging at the start of eukaryogenesis. (shrink)

In this paper it is argued that certain stimulus-response learning models which are adequate to represent finite automata (acceptors) are not adequate to represent noninitial state input-output automata (transducers). This circumstance suggests the question whether or not the behavior of animals if satisfactorily modelled by automata is predictive. It is argued in partial answer that there are automata which can be explained in the sense that their transition and output functions can be described (roughly, Hempel-type covering law explanation) while their (...) behaviors are in principle not predictable short of possession of their complete histories or of information concerning present internal states by indirect observation. (shrink)

Despite thermodynamic, bioenergetic and phylogenetic failings, the 81‐year‐old concept of primordial soup remains central to mainstream thinking on the origin of life. But soup is homogeneous in pH and redox potential, and so has no capacity for energy coupling by chemiosmosis. Thermodynamic constraints make chemiosmosis strictly necessary for carbon and energy metabolism in all free‐living chemotrophs, and presumably the first free‐living cells too. Proton gradients form naturally at alkaline hydrothermal vents and are viewed as central to the origin of life. (...) Here we consider how the earliest cells might have harnessed a geochemically created proton‐motive force and then learned to make their own, a transition that was necessary for their escape from the vents. Synthesis of ATP by chemiosmosis today involves generation of an ion gradient by means of vectorial electron transfer from a donor to an acceptor. We argue that the first donor was hydrogen and the first acceptor CO2. (shrink)

Leonid Levin showed that every algorithm computing a function has an optimal inverter. Recently, we applied his result in various contexts: existence of optimal acceptors, existence of hard sequences for algorithms and proof systems, proofs of Gödel’s incompleteness theorems, analysis of the complexity of the clique problem assuming the nonuniform Exponential Time Hypothesis. We present all these applications here. Even though a simple diagonalization yields Levin’s result, we believe that it is worthwhile to be aware of the explicit result. The (...) purpose of this survey is to convince the reader of our view. (shrink)

tRNA nucleotidyltransferase adds the invariant CCA‐terminus to the tRNA 3′‐end, a central step in tRNA maturation. This CCA‐adding enzyme is a specialized RNA polymerase that synthesizes the CCA sequence at high fidelity in all kingdoms of life. Recently, an additional function of this enzyme was identified, where it generates a specific degradation tag on structurally unstable tRNAs. This tag consists of an additional repeat of the CCA triplet, leading to a 3′‐terminal CCACCA sequence. In order to explain how the enzyme (...) catalyzes this extended polymerization reaction, Kuhn et al. solved a series of co‐crystal structures of the CCA‐adding enzyme from Archaeoglobus fulgidus in complex with different tRNA substrates. They show that the enzyme forces a bound unstable tRNA to refold the acceptor stem for a second round of CCA‐addition, while stable transcripts are robust enough to resist this isomerization. In this review, we discuss how the CCA‐adding enzyme uses a simple yet very elegant way to scrutinize its substrates for sufficient structural stability and, consequently, functionality. (shrink)

The three essays presented here concern natural connections between grammatical derivations and structures provided by certain standard grammar formalisms, on the one hand, and deductions in logical systems, on the other hand. In the first essay we analyse the adequacy of Polish notation for higher-order languages. The Ajdukiewicz algorithm (Ajdukiewicz 1935) is discussed in terms of generalized MP-deductions. We exhibit a failure in Ajdukiewicz’s original version of the algorithm and give a correct one; we prove that generalized MP-deductions have the (...) frontier property, which is essential for the plausibility of Polish notation. The second essay deals with logical systems corresponding to different grammar formalisms, as e.g. Finite State Acceptors, Context-Free Grammars, Categorial Grammars, and others. We show how can logical methods be used to establish certain linguistically significant properties of formal grammars. The third essay discusses the interplay between Natural Deduction proofs in grammar oriented logics and semantic structures expressible by typed lambda terms and combinators. (shrink)

Appeals to the moral authority of nature play an important role in ethical discussions about the acceptability of prenatal testing. While opponents consider testing a dangerous violation of the moral inviolable course of nature, defenders see testing as a new step in improving dominion over nature. In this study we explored the meaning of appeals to nature among pregnant women to whom a prenatal screening test was offered and the impact of these appeals on their choices regarding the acceptance of (...) screening. Contrary to theoretical debates we found that appeals to the moral authority of nature do not prevent women from welcoming the possibilities of controlling the outcomes of pregnancy, neither do they provide prima facie justification for (not) intervening in the natural course of events. Both acceptors and decliners believed in an inherent morality in nature that must be respected. They welcome the possibility of knowing more about the health of the fetus and to make their own reproductive decisions. Concerns for the quality of their child's life and for their capacity to assure a good life for their family and disabled child appear to play a central role in the decision regarding the use of screening. Appeals to nature can be interpreted as an attempt to justify beliefs regarding suffering that must be avoided and the impact that family interests may have on the decision. These findings have significant implications for ethical guidance in debates about the acceptability and boundaries of control of offspring characteristics by prenatal testing. (shrink)

Electron redistribution is the cornerstone of our understanding of chemical reactivity. For the vast majority of organic reactions electrons are assumed to move in pairs providing explanatory mechanisms through the generation of intermediate structures. But for many transformations these discrete steps are idealized constructs, involving intermediates assumed but not empirically justified. This unitary perspective predicated on the curved arrow formalism has resulted in the scenario where for many organic transformations our supposed understanding far surpasses our growing knowledge. Reformulating organic mechanisms (...) to include single electron transfer as a component of, or an alternative to, the prevailing iconic descriptions can provide for a more empirically adequate mechanistic description. In addition using the language of SET presents an opportunity to unify mechanistic concepts under a common donor/acceptor framework. (shrink)

The fifth base in human DNA, 5‐methylcytosine, is inherently mutagenic. This has led to marked changes in the distribution of the CpG methyl acceptor site and an 80% depletion in its frequency of occurrence in vertebrate DNA. The coding regions of many genes contain CpGs which are methylated in sperm and serve as hot spots for mutation in human genetic diseases. Fully 30–40% of all human germline point mutations are thought to be methylation induced even though the CpG dinucleotide (...) is under‐represented and efficient cellular repair systems exist. Importantly, tumor suppressor genes such as p53 also contain methylated CpGs and these serve as hot spots for mutations in some, but not all, human cancers. Comparison of the spectrum of mutations present in this gene in different human cancers allows for predictions to be made on the molecular mechanisms of tumorigenesis. (shrink)

Biodiesel was produced by transesterification of waste olive oil with methanol and Novozym®435. The effect of the molar ratio of methanol to triolein, mode of methanol addition, reaction temperature, and mixing speed on biodiesel yield was determined. The effect of different acyl acceptors and/or solvents on biodiesel yield was also evaluated. Finally, the efficacy of Novozym®435 was determined by reusing the enzyme after washing it with a solvent.

The lateral mobility of membrane lipids and proteins is presumed to play an important functional role in biomembranes. Photobleaching studies have shown that many proteins in the plasma membrane have diffusion coefficients at least an order of magnitude lower than those obtained when the same proteins are reconstituted in artificial bilayer membranes. Depending on the protein, it has been shown that either the cytoplasmic domain or the ectodomain is the key determinant of its lateral mobility. Single particle tracking microscopy, (...) which allows the motions of single or small groups of membrane molecules to be followed, promises not only to reveal new features of membrane dynamics, but also to help explain longstanding puzzles presented by the photobleaching studies, particularly the so‐called immobile fraction. The combination of the two complementary technologies should measurably enhance our understanding of membrane microstructure. (shrink)

During the last decade neurotransplantation has developed into a technique with the possible potential to repair damaged or degenerating human brain. Effective neurotransplantation has so far been based on the use of fetal brain tissue derived from aborted embryos or fetuses. The ethical issues related to this new therapeutic approach therefore not only concern the possible adverse side effects for a neural graft-receiving patient, but also the relationship between the requirements for fetal tissue and the decision-making process for induced abortion. (...) Although for decades human embryos and fetuses have been the subject of biomedical studies, and, in principle, their use has therefore not been seen as ethically objectionable, the above points made it necessary to reconsider the moral issues. The present paper points out several of these issues, both from the donor and acceptor (patient) point of view. The conclusion is that under a series of restrictions intended to prevent the use of grafts from encouraging induced abortions and to maintain high standards of respect for life and human dignity, neurotransplantation using embryonic or fetal brain tissue parts cannot be rejected on moral grounds. (shrink)

Presented in this study is an analysis of the electronic properties of doped diamond calculated using the Vienna ab initio simulation package, employing density functional theory within the generalized-gradient approximation. The dopants studied here have been inserted substitutionally into a 64-atom diamond supercell and include the single-electron acceptors boron and aluminium, the single-electron donors nitrogen and phosphorus and the double-electron donors oxygen and sulphur. Co-doping of diamond with sulphur and boron has also been briefly examined. The doped supercells have been (...) relaxed, followed by calculation of electronic properties from the electronic density of states such as the indirect bandgap E g , the valence bandwidth and an examination of the acceptor and donor states in the bandgap. It is anticipated that this study will provide a useful comparison of the third- and fourth-row donors and acceptors in diamond. (shrink)

In this study an analysis is presented of the bonding and structural properties of dehydrogenated and hydrogenated doped cylindrical diamond nanowires calculated using the Vienna Ab Initio Simulation Package, employing density functional theory within the generalized-gradient approximation. The dopants studied here have been inserted substitutionally along the axis of an infinite one-dimensional diamond nanowire and include the single-electron acceptor boron and the single-electron donor nitrogen. The doped nanowires have then been re-relaxed, and properties compared with the undoped structures. The (...) structural properties of relaxed nanowires considered here include an examination bonding via the electron charge density, with the aim of providing a better understanding of the effects of dopants on the stability of diamond nanostructures and nanodevices. (shrink)

— We prescnt here a comparative study on the decay of chlorophyll a fluorescence yield in thylakoid membranes and photosystem 11 ‐enriched samples, measured with multifrequency cross‐correlation phase fluorometry. These measurements confirm the general conclusions of Van Mieghem ef al., obtained with a flash method, on the effects of reduction of the primary quinone acceptor on ChI a fluorescence yield of PSI. Different states of the reaction centers of PSII were produced by: pretreatment with sodium dithionite and mcthyl viologen (...) followed by laser illumination: the doubly reduced QA centers: with laser illumination or pretreatment with diuron: QA‐ centers; and the addition of micromolar concentration of dichlorobenzoquinonc : oxidized QA centers. The data were analyzed with Lorentzian distribution as well as with multiexponential fluorescence decay functions. The analysis with Lorcntzian distribution function showed that upon formation of QA. the major lifetime distribution peak shifted to longer lifetimes: from 0.25 ns to 1.66 ns and from 0.24 ns to 1.31 ns. However, when QAHZ was formed, the lifetime distribution peaks shifted back to shorter lifetimes both in thylakoids and PSIl membranes. Multiexponential analysis showed three lifetime components: fast, middle and slow. When QA was formed in PS11 centers, the amplitude of the fast Component decreased, but both thc amplitude and the lifetime of the middle component increased severalfold. However, when QA was doubly reduccd, the amplitude of the fast component increased and the amplitude of the middle component decreased: in addition, the lifetime of the slow component increased. All of the above results are consistent with the conclusions that PS11 charge separation is decreased when QA is formed and increased when doubly reduced QA is formed. Copyright © 1993, Wiley Blackwell. All rights reserved. (shrink)

Various lipid transfer proteins (LTPs) mediate the inter‐organelle transport of lipids. By working at membrane contact zones between donor and acceptor organelles, LTPs achieve rapid and accurate inter‐organelle transfer of lipids. This article will describe the emerging paradigm that the action of LTPs at organelle contact zones generates metabolic channeling events in lipid metabolism, mainly referring to how ceramide synthesized in the endoplasmic reticulum is preferentially metabolized to sphingomyelin in the distal Golgi region, how cholesterol and phospholipids receive specific (...) metabolic reactions in mitochondria, and how the hijacking of host LTPs by intracellular pathogens may generate new channeling‐like events. In addition, the article will discuss how the function of LTPs is regulated, exemplified by a few representative LTP systems, and will briefly touch on experiments that will be necessary to establish the paradigm that LTP‐mediated inter‐organelle transport of lipids is one of the mechanisms of compartmentalization‐based metabolic channeling events. (shrink)

Introduction of O2 to Earth's early biosphere stimulated remarkable evolutionary adaptations, and a wide range of electron acceptors allowed diverse, energy-yielding metabolic pathways. Enzymatic reduction of O2 yielded a several-fold increase in energy production, enabling evolution of multi-cellular animal life. However, utilization of O2 also presented major challenges as O2 and many of its derived reactive oxygen species are highly toxic, possibly impeding multicellular evolution after the Great Oxidation Event. Remarkably, ROS, and especially hydrogen peroxide, seem to play a major (...) part in early diversification and further development of cellular respiration and other oxygenic pathways, thus becoming an intricate part of evolution of complex life. Hence, although harnessing of chemical and thermo-dynamic properties of O2 for aerobic metabolism is generally considered to be an evolutionary milestone, the ability to use ROS for cell signaling and regulation may have been the first true breakthrough in development of complex life. The importance of Earth's oxygen cycle and reactive oxygen species produced on the evolution of complex life. The ability to detoxify oxygen species is not a trait that emerged as a response to increased O2 levels but rather crucial adaptation to the early Earth's weakly oxic microenvironments. (shrink)

Objective The study evaluated patient informed consent (IC) for the Quad screen and examined differences in IC between test acceptors and test refusers. A multidimensional model of IC was used. Methods Women seeking prenatal care at nine obstetrics clinics in a large Midwestern city completed surveys between February and December 2006. Surveys contained measures for three dimensions of IC: intention, understanding and controlling influence. Results 56.2% of women did not meet criteria for all three of our dimensions of IC and (...) therefore failed to give it. The failure rate was higher among women who choose to screen (72.6%) than women who choose not screen (50%) (p < 0.001). Women who met all criteria for IC were over three times less likley to choose to screen (or = 0.32, CI 0.17–0.62 ( p < 0.01)) than women who did not meet criteria for IC. Conclusion The decision to screen for fetal anomalies is less of a deliberated action than the decision not to screen. Women who lack a fundamental understanding of the purpose and nature of the screen may be operating on the belief that the screen is part of standard care and presents no need to deliberate. (shrink)

A decade ago I postulated that ROS formation in mitochondria was influenced by different FADH2/NADH (F/N) ratios of catabolic substrates. Thus, fatty acid oxidation (FAO) would give higher ROS formation than glucose oxidation. Both the emergence of peroxisomes and neurons not using FAO, could be explained thus. ROS formation in NADH:ubiquinone oxidoreductase (Complex I) comes about by reverse electron transport (RET) due to high QH2 levels, and scarcity of its electron‐acceptor (Q) during FAO. The then new, unexpected, finding of (...) an FAO enzyme, ACAD9, being involved in complex I biogenesis, hinted at connections in line with the hypothesis. Recent findings about ACAD9's role in regulation of respiration fit with predictions the model makes: cementing connections between ROS production and F/N ratios. I describe how ACAD9 might be central to reversing the oxidative damage in complex I resulting from FAO. This seems to involve two distinct, but intimately connected, ACAD9 characteristics: (i) its upregulation of complex I biogenesis, and (ii) releasing FADH2, with possible conversion into FMN, the crucial prosthetic group of complex I. Also see the video abstract here: https://youtu.be/N7AT_HBNumg. (shrink)

The performance of population programmes suggests that even the more successful ones have acceptance rates that are only a fifth or so of what is needed to reduce population growth rates to the 1% a year that permits significant improvements in income per head. It is also clear that no programmes use bonuses at anything approaching the monetary values per recipient that are justifiable. There is no guarantee that such bonuses will produce the number of acceptors that are needed, but (...) it is fairly evident that without larger bonuses there will not be enough acceptors. (shrink)

The Second Taiwan IUD Follow-up Survey, reported on here, is a representative sample of all IUD acceptors in Taiwan up to the middle of 1966. The data show that 30 months after insertion, 36% of acceptors are continuing users, on a first segment basis, and that if reinsertions are taken into account, the proportion increases to 45%. Compared with extensive clinic data from the city of Taichung, the island-wide termination rates are about 5% higher at 30 months, for first segments, (...) but the all segment rates show little difference at this duration. (shrink)

Despite thermodynamic, bioenergetic and phylogenetic failings, the 81‐year‐old concept of primordial soup remains central to mainstream thinking on the origin of life. But soup is homogeneous in pH and redox potential, and so has no capacity for energy coupling by chemiosmosis. Thermodynamic constraints make chemiosmosis strictly necessary for carbon and energy metabolism in all free‐living chemotrophs, and presumably the first free‐living cells too. Proton gradients form naturally at alkaline hydrothermal vents and are viewed as central to the origin of life. (...) Here we consider how the earliest cells might have harnessed a geochemically created proton‐motive force and then learned to make their own, a transition that was necessary for their escape from the vents. Synthesis of ATP by chemiosmosis today involves generation of an ion gradient by means of vectorial electron transfer from a donor to an acceptor. We argue that the first donor was hydrogen and the first acceptor CO2. (shrink)

This study was conducted from May to June 2005 to describe the demographic characteristics and factors that affect the VCT acceptance as well as the HIV prevalence among youth VCT acceptors in Addis Ababa. Both quantitative and qualitative methods of data collection were employed. The quantitative data was generated from a two years (October 2002 to December 2004) VCT service utilization data obtained from four youth centers located in Addis Ababa. The data was analysed using univariate and multivariate analysis and (...) results were presented in the form of text, table and figures. The significance and level of associations between independent and dependant variables was computed using X2 tests and odds ratios with 95% confidence interval. The qualitative data was presented in a form of narrations using excerpts. During the period a total of 3220, youth age 15 - 24 years have sought VCT service from the four youth centres VCT service outlets. Higher proportions of females (60.2%) and youth in the age category of 20 – 24 years (58.0%) have accessed the service. The major reasons given for seeking VCT were found to be similar for male and female acceptors. About 71.2% said that they sought for VCT service because they wanted to know their HIV status, 9.5% reported because they were suspicious of being infected with HIV and 5.0% tested because they wanted to go aboard. The HIV prevalence rate is found to be significantly higher, (p = 0.00), among females 9.2 % than in males 1.7 %. Gender segregated multivariate analysis of risk factors indicated significant association of HIV infection with lack of formal education for both males and female: for males P= 0.045, OR = 18.3, 95% CI: 1.1 – 317.7 and for females P=0.00, OR=19.4, 95 % CI: 5.1 – 73.8. Additionally for females HIV infection is markedly associated with being widowed and commercial sex worker. The most common barrier for youth in Addis from accessing VCT were found to be: fear of being emotionally affected if found out HIV positive, stigma and discrimination, lack of money to pay for VCT service, absence of care and support services for people who are already infected. The challenges encountered by youth VCT counsellors were stress, absence of medical care for PLWHA, inadequacy of budget, lack of counsellors and supportive supervision. This study showed high burden of HIV infection among female youth and the risk factors associated with it. Formal education for both females and males is strongly recommended. Female focused HIV prevention interventions are indicated. Additional recommendations are also made in relation to barriers to access youth VCT and for the challenges encountered by youth VCT service providers. (shrink)

Phototoxicity frequently occurs during live fluorescence microscopy, and its consequences are often underestimated. Damage to cellular macromolecules upon excitation light illumination can impair sample physiology, and even lead to sample death. In this review, we explain how phototoxicity influences live samples, and we highlight that, besides the obvious effects of phototoxicity, there are often subtler consequences of illumination that are imperceptible when only the morphology of samples is examined. Such less apparent manifestations of phototoxicity are equally problematic, and can change (...) the conclusions drawn from an experiment. Thus, limiting phototoxicity is a prerequisite for obtaining reproducible quantitative data on biological processes. We present strategies to reduce phototoxicity, e.g. limiting the illumination to the focal plane and suggest controls for phototoxicity effects. Overall, we argue that phototoxicity needs increased attention from researchers when designing experiments, and when evaluating research findings. (shrink)