Efficient Blind Spectral Unmixing of Fluorescently Labeled Samples Using Multi-Layer Non-Negative Matrix Factorization

The ample variety of labeling dyes and staining methods available in fluorescence microscopy has enabled biologists to advance in the understanding of living organisms at cellular and molecular level. When two or more fluorescent dyes are used in the same preparation, or one dye is used in the presence of autofluorescence, the separation of the fluorescent emissions can become problematic. Various approaches have been recently proposed to solve this problem. Among them, blind non-negative matrix factorization is gaining interest since it requires little assumptions about the spectra and concentration of the fluorochromes. In this paper, we propose a novel algorithm for blind spectral separation that addresses some of the shortcomings of existing solutions: namely, their dependency on the initialization and their slow convergence. We apply this new algorithm to two relevant problems in fluorescence microscopy: autofluorescence elimination and spectral unmixing of multi-labeled samples. Our results show that our new algorithm performs well when compared with the state-of-the-art approaches for a much faster implementation.     

Coping with multiple enemies: an integration of molecular and ecological perspectives

How plants respond to attack by the range of herbivores and pathogens that confront them in the field is the subject of considerable research by both molecular biologists and ecologists. However, in spite of the shared focus of these two bodies of research, there has been little integration between them. We consider the scope for such integration, and how greater dialogue between molecular biologists and ecologists could advance understanding of plant responses to multiple enemies.     

Report from the President the Biological Stain Commission: Its Goals, Its Past and Its Present Status

This is a brief overview of the goals, evolution, and present status of the Biological Stain Commission. The main function of the Commission is the testing and certification of dye batches intended for biological applications. The testing is supported by charges made for batch testing and by the sale of certification labels affixed to individual dye containers. Submission of dyes for testing is voluntary, depending on the cooperation of the companies who sell them and the consumers who buy them. The supportive role of the University of Rochester School of Medicine and Dentistry—both past and present—is not well known and should be. Increasingly federal regulations affect the production, availability, and cost of dyes. Commission income from the sale of labels has decreased in recent years. Continuation of its work requires changes that will produce more income. Much dye is now sold in solutions instead of dry powders. The value of using Stain Commission certified dyes whenever possible is illustrated by the case of basic fuchsin. Years ago this dye was a mixture. Most basic fuchsin now marketed consists mainly of either pararosanilin (Colour Index No. 42500) or rosanilin (C.I. No. 42510). The Biological Stain Commission discovered that some certified batches of both pararosanilin and rosanilin sold as “basic fuchsin” had incorrect C.I. numbers on the labels. Sometimes that caused failure of the aldehyde fuchsin stain. Unless made with pararosanilin, aldehyde fuchsin does not stain pancreatic islet B-cells, elastic fibers, and hepatitis B surface antigen in unoxidized sections. Mislabelling by packagers may interfere with other applications of pararosanilin and rosanilin. The Commission acted to publicize and correct this problem. Biological Stain Commission publications help educate microscopists and histotechnologists about dyes and their best use. Stain Commission representatives from member scientific societies provide valuable input about changes in the availability and quality of such dyes as hematoxylin and others; they also provide useful feedback to their societies about dye problems. Each new generation of biologists and histotechnologists should be taught the importance of using only Stain Commission certified stains when available. They should be taught also to notify the Stain Commission whenever they experience problems with any certified dye.     

Comparison of historic Grübler dyes with modern counterparts using thin layer chromatography.

The aniline dye industry was created in 1856 when William Perkin prepared the dye, mauve, from coal tar. Following that discovery, several dye manufacturing businesses were formed in Western Europe, most successfully in Germany. It was to these companies that early investigators turned to obtain these new dyes for the developing field of biology. In 1880, Dr. Georg Grübler started a company in Germany to supply the needs of biologists. Grübler dyes developed a reputation for excellence. In the study reported here, 29 samples of 12 Grübler dyes were compared to modern counterparts using thin layer chromatography. The dyes studied were basic fuchsine, acid fuchsine, safranine, pyronine, aniline blue, ponceau, gentian violet, methylene blue, orange G, malachite green, and Sudan III and IV. I found that these early Grübler dyes closely resembled modern day counterparts; however, the use of synonyms was confusing and some of the fat stains were mislabeled by modern criteria. The chromatograms of some dyes exhibited smearing, probably representing multiple closely related dye species. The study of old dyes provides interesting comparisons with modern counterparts as the center of dye manufacturing is moving from Europe and the United States to Asia.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

Cortex shatters the glass ceiling

Recreating developmental structures in vitro has been a primary challenge for stem cell biologists. Recent studies in Cell Stem Cell (Eiraku et al., 2008) and Nature (Gaspard et al., 2008) demonstrate that embryonic stem cells can recapitulate early cortical development, enabling them to generate specific cortical subtypes.     

Comparison of historic Grübler dyes with modern counterparts using thin layer chromatography

The aniline dye industry was created in 1856 when William Perkin prepared the dye, mauve, from coal tar. Following that discovery, several dye manufacturing businesses were formed in Western Europe, most successfully in Germany. It was to these companies that early investigators turned to obtain these new dyes for the developing field of biology. In 1880, Dr. Georg Grübler started a company in Germany to supply the needs of biologists. Grübler dyes developed a reputation for excellence. In the study reported here, 29 samples of 12 Grübler dyes were compared to modern counterparts using thin layer chromatography. The dyes studied were basic fuchsine, acid fuchsine, safranine, pyronine, aniline blue, ponceau, gentian violet, methylene blue, orange G, malachite green, and Sudan III and IV. I found that these early Grübler dyes closely resembled modern day counterparts; however, the use of synonyms was confusing and some of the fat stains were mislabeled by modern criteria. The chromatograms of some dyes exhibited smearing, probably representing multiple closely related dye species. The study of old dyes provides interesting comparisons with modern counterparts as the center of dye manufacturing is moving from Europe and the United States to Asia.     

The threat from creationism to the rational teaching of biology

Most biologists outside the USA and a few other countries, like Australia and Canada, are under the impression that the threat to the teaching of biology represented by creationism does not concern them directly. This is unfortunately no longer true: the recent growth of creationism, especially in its pseudo-scientific manifestation known as "intelligent design", has been obvious in several countries of Western Europe, especially the UK, Germany and Poland, and it is beginning to be noticeable in Brazil, and maybe elsewhere in Latin America. The problem is complicated by the fact that there are not just two possibilities, evolution and creationism, because creationism comes in various incompatible varieties. Turkey is now a major source of creationist propaganda outside the USA, and is especially significant in relation to its influence on Muslim populations in Europe. The time for biologists to address the creationist threat is now.     

Syntheses and DNA-binding studies of a series of unsymmetrical cyanine dyes: structural influence on the degree of minor groove binding to natural DNA

Twelve crescent-shaped unsymmetrical dyes have been synthesized and their interactions with DNA have been investigated by spectroscopic methods. A new facile synthetic route to this type of cyanine dyes has been developed, involving the preparation of 6-substituted 2-thiomethyl-benzothiazoles in good yields. The new dyes are analogues to the minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a 6-methylbenzothiazole substituent. Herein we further investigate the role of the extending benzazole heterocycle, as well as of the pyridine or quinoline moiety of the cyanine chromophore, for the binding mode of these crescent-shaped dyes to calf thymus DNA. Flow LD and CD studies of the 12 dyes show that the extent of minor groove binding to mixed sequence DNA varies significantly between the dyes. We find that hydrophobicity and size are the crucial parameters for recognition of the minor groove. The relatively high fluorescence quantum yield of many of these cyanines bound to DNA, combined with their absorption at long wavelengths, may render them useful in biological applications. In particular, two of the benzoxazole containing dyes BOXTO and 2-BOXTO show a high degree of minor groove binding and quantum yields of 0.52 and 0.32, respectively, when bound to DNA.     

Basic and applied aspects in the microbial degradation of azo dyes

Azo dyes are the most important group of synthetic colorants. They are generally considered as xenobiotic compounds that are very recalcitrant against biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several microorganisms are able, under certain environmental conditions, to transform azo dyes to non-colored products or even to completely mineralize them. Thus, various lignolytic fungi were shown to decolorize azo dyes using ligninases, manganese peroxidases or laccases. For some model dyes, the degradative pathways have been investigated and a true mineralization to carbon dioxide has been shown. The bacterial metabolism of azo dyes is initiated in most cases by a reductive cleavage of the azo bond, which results in the formation of (usually colorless) amines. These reductive processes have been described for some aerobic bacteria, which can grow with (rather simple) azo compounds. These specifically adapted microorganisms synthesize true azoreductases, which reductively cleave the azo group in the presence of molecular oxygen. Much more common is the reductive cleavage of azo dyes under anaerobic conditions. These reactions usually occur with rather low specific activities but are extremely unspecific with regard to the organisms involved and the dyes converted. In these unspecific anaerobic processes, low-molecular weight redox mediators (e.g. flavins or quinones) which are enzymatically reduced by the cells (or chemically by bulk reductants in the environment) are very often involved. These reduced mediator compounds reduce the azo group in a purely chemical reaction. The (sulfonated) amines that are formed in the course of these reactions may be degraded aerobically. Therefore, several (laboratory-scale) continuous anaerobic/aerobic processes for the treatment of wastewaters containing azo dyes have recently been described.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

Reverse engineering the embryo: a graduate course in developmental biology for engineering students at the University of Manitoba,Canada

Our desire to educate engineers to be able to understand the component processes of embryogenesis, is driven by the notion that only when principles borrowed from mathematics, fluid mechanics, materials science, etc. are applied to classical problems in developmental biology, will sufficient comprehension be achieved to permit successful understanding and therapeutic manipulation of embryos. As it now stands, biologists seldom possess either skills or interest in those areas of endeavor. Thus, we have determined that it is easier to educate engineers in the principles of developmental biology than to help biologists deal with the complexities of engineering. We describe a graduate course that has been taken, between 1999 and 2002, by 17 engineering students. Our goal is to prepare them to reverse engineer the embryo, i.e., to look at it as an object or process whose construction, albeit self-construction, might be explicable in terms of engineering principles applied at molecular, cellular and whole embryo levels.     

Logical puzzles and scientific controversies: The nature of species,viruses and living organisms

In the past, biologists believed that species were stable and permanent entities and they viewed them as natural kinds which, like the chemical elements, exist in nature independently of any human conceptualization. After Darwin, biologists came to accept that species were the products of evolution and natural selection and were not immutable natural kinds.     

The Virtual Cell: a software environment for computational cell biology

The newly emerging field of computational cell biology requires software tools that address the needs of a broad community of scientists. Cell biological processes are controlled by an interacting set of biochemical and electrophysiological events that are distributed within complex cellular structures. Computational modeling is familiar to researchers in fields such as molecular structure, neurobiology and metabolic pathway engineering, and is rapidly emerging in the area of gene expression. Although some of these established modeling approaches can be adapted to address problems of interest to cell biologists, relatively few software development efforts have been directed at the field as a whole. The Virtual Cell is a computational environment designed for cell biologists as well as for mathematical biologists and bioengineers. It serves to aid the construction of cell biological models and the generation of simulations from them. The system enables the formulation of both compartmental and spatial models, the latter with either idealized or experimentally derived geometries of one, two or three dimensions.     

Biodiversity,biological uncertainty,and setting conservation priorities

In a world of massive extinctions where not all taxa can be saved, how ought biologists to decide their preservation priorities? When biologists make recommendations regarding conservation, should their analyses be based on scientific criteria, on public or lay criteria, on economic or some other criteria? As a first step in answering this question, we examine the issue of whether biologists ought to try to save the endangered Florida panther, a well known “glamour” taxon. To evaluate the merits of panther preservation, we examine three important arguments of biologists who are skeptical about the desirability of panther preservation. These arguments are (1) that conservation dollars ought to be spent in more efficient ways than panther preservation; (2) that biologists and conservationists ought to work to preserve species before subspecies; and (3) that biologists and conservationists ought to work to save habitats before species or subspecies. We conclude that, although all three arguments are persuasive, none of them provides convincing grounds for foregoing panther preservation in favor of other, more scientifically significant conservation efforts. Our conclusion is based, in part, on the argument that biologists ought to employ ethical, as well as scientific, rationality in setting conservation priorities and that ethical rationality may provide persuasive grounds for preserving taxa that often are not viewed by biologists as of great importance.     

Assessing the prospects for a return of organisms in evolutionary biology

An argument has been raised from various perspectives against the Modern Synthesis (MS) in the past two decades: it has forgotten organisms. Niche construction theorists (Odling-Smee et al. 2003), developmental biologists like West-Eberhard (2003) and Evo-Devo elaborated various views which concur on a rehabilitation of the explanatory role of organisms, formerly neglected by an evolutionary science mostly centered on genes. This paper aims at assessing such criticisms by unraveling the specific arguments they use and evaluating how empirical findings may support them. In the first section, I review the usual critiques about the way MS treats organisms and show that the organisms-concerned critique is multifaceted, and I use the controversy about units of selection in order to show that purely conceptual and empirical arguments have been mixed up when organisms were concerned. In the second section, I consider successively the challenges raised to evolutionary MS by structuralist biologists and then the developmentalist challenge mostly raised by Evo-Devo. I distinguish what is purely conceptual among those criticisms and what mostly relies on recent empirical findings about genome activation, inheritance, and epigenetics. The last section discusses another program in MS, namely "evolutionary transitions" research, as enquiry into the emergence of organisms.     

Effects of differently hydrophobic solvents on the aggregation of cationic dyes as measured by quenching of fluorescence and/or metachromasia of the dyes

Aggregation of some cationic dyes leads to the appearance of their metachromatic spectra and/or quenching of their fluorescence. Ethanol and urea destroy metachromasia and enhance the fluorescence of such dyes by disaggregating them, suggesting hydrophobic bonds to be involved in their aggregation as in the formation of soap micelles or globular proteins. The ability of alcohols to disaggregate cationic dyes has been shown to be increased in the series methanol, ethanol, iospropanol and tertiary-butanol which is the order of increasing hydrophobic character of the alcohols themselves. Dimethyl urea is shown to be more effective than urea in destroying the metachromasia of toluidine blue, thus supporting the idea of hydrophobic bond to be involved in dye aggregation. Rhodamine 6 G undergoes quenching of its fluorescence in presence of a suitable polyanion but it is not metachromatic like acridine orange. Since only some specific cationic dyes undergo spectral changes with aggregation such changes seem to be the secondary effects of aggregation.Pool Officer, Scientists' Pool, Council of Scientific & Industrial Research (India), attached to the Bose Institute.     

One cell, two cell, red cell, blue cell: The persistence of a unicellular stage in multicellular life histories

As developmental biologists come closer to understanding at the molecular and genetic levels how a zygote becomes an adult, it is easy to forget that the very phenomenon that gives them an occupation remains a vexing problem to evolutionary biologists: why do unicellular stages persist in life histories of multicellular organisms? There are two explanatory hypotheses. One is that a unicellular stage purges multicellular organisms of deleterious mutations by exposing offspring that are each uniformly of one genotype to selection. Another is that a one-cell stage reduces conflicts of interest among genetically different replicators within an organism.     

Benzidine-based dyes: effects of industrial practices,regulations, and world trade on the biological stains market

One of the most sweeping changes in the dye industry since the advent of synthetic dyes grew out of the health risks associated with benzidine. Dyes made from benzidine and its derivatives were used around the world until adverse health effects become incontrovertible. Workers and family members of workers involved in production and use of benzidine-based dyes had a high incidence of bladder cancer. Following publication of several reports documenting this health hazard, dye makers in the USA, Europe, and Japan phased these dyes out of production in the 1970s. Government regulations lent legal support for these voluntary initiatives. Two strategies subsequently evolved to compensate: developed nations brought alternative substances to market while emerging countries increased production of carcinogenic dyes and sold them at discount prices around the world. Nearly all dye manufacturing now has moved away from nations whose costs of production and compliance rendered them unable to compete. The purpose of this brief review is to publicize the health risks associated with dyes made from benzidine and its congeners, and to alert all companies and end users handling these dyes for biomedical applications that composition of the product and lot-to-lot variability may be problematic because of the manufacturing and distribution practices of the countries where they are produced.