Colorimetry for the Stain Technologist. I. The Specification of Color

This paper describes color specification for the stain technologist. The principles of color stimulus specification are reviewed in terms of the conventions of the Commission Internationale de l'Eclairage (CIE). The text is largely self-contained and has been written so that it can be understood easily by a reader with no prior knowledge of color science. The paper starts with definitions of color and related psychological, psychophysical and colori-metric terms. X, Y, Z color space is described. It is shown that any color stimulus may be unambiguously defined in terms of a set of three numbers. The CIE 1931 Chromaticity Diagram is described. Worked examples are given for the calculation of tristimulus values and chromaticity coordinates using three different illuminants. The usefulness of color specification is illustrated by a number of examples using Romanowsky stained blood cells or Papanicolaou stained epithelial cells from the uterine cervix.     

Colorimetry for the stain technologist. I. The specification of color

This paper describes color specification for the stain technologist. The principles of color stimulus specification are reviewed in terms of the conventions of the Commission Internationale de l'Eclairage (CIE). The text is largely self-contained and has been written so that it can be understood easily by a reader with no prior knowledge of color science. The paper starts with definitions of color and related psychological, psychophysical and colorimetric terms. X, Y, Z color space is described. It is shown that any color stimulus may be unambiguously defined in terms of a set of three numbers. The CIE 1931 Chromaticity Diagram is described. Worked examples are given for the calculation of tristimulus values and chromaticity coordinates using three different illuminants. The usefulness of color specification is illustrated by a number of examples using Romanowsky stained blood cells or Papanicolaou stained epithelial cells from the uterine cervix.     

On the trichromatic and opponent-process theories: an article by E. Schr?dinger

A translation of Schr?dinger's paper, 'On the relation of the tetrachromatic theory to the trichromatic theory' (1925), is accompanied by a commentary. Schr?dinger applies a projective transformation to a standard chromaticity diagram, to demonstrate the common geometry of the chromaticity diagrams derived from the trichromatic and opponent-process theories of color vision.     

The colour hexagon: a chromaticity diagram based on photoreceptor excitations as a generalized representation of colour opponency

Summary A chromaticity diagram which plots the 3 photoreceptor excitations of trichromatic colour vision systems at an angle of 120° is presented. It takes into acount the nonlinear transduction process in the receptors. The resulting diagram has the outline of an equilateral hexagon. It is demonstrated by geometrical means that excitation values for any type of spectrally opponent mechanism can be read from this diagram if the weighting factors of this mechanism add up to zero. Thus, it may also be regarded as a general representation of colour opponent relations, linking graphically the Young-Helmholtz theory of trichromacy and Hering's concept of opponent colours. It is shown on a geometrical. basis that chromaticity can be coded unequivocally by any two combined spectrally opponent mechanisms, the main difference between particular mechanisms being the extension and compression of certain spectral areas. This type of graphical representation can qualitatively explain the Bezold-Brücke phenomenon. Furthermore, colour hexagon distances may be taken as standardized perceptual colour distance values for trichromatic insects, as is demonstrated by comparison with behavioural colour discrimination data of 3 hymenopteran species.     

Colorimetry for the Stain Technologist. III. The Specification of Color Difference

This paper illustrates the calculation of color differences, involving luminance as well as chromaticity components. Color differences have been calculated for a large number of stained histological objects. Four different color difference formulae have been used, namely, those associated with the FMC 2, U*V*W*, L*u*v* and L^*a^*b^* systems. Comparison has first been made between various hematological substrates after staining with two different azure B-eosin Y stains. Next, comparison is made for the same substrates after staining with one of the azure B stains and a methylene blue-eosin Y stain. Pairwise comparison is also made of various substrates from the epithelium of the uterine cervix after Papanicolaou staining. Finally, pairwise comparison documents color differences accompanying maturation for the erythroid and myeloid cell lines in azure B-eosin Y stained bone marrow. The limitations of current color difference formulae are discussed.     

Similarity transformation approach to identifiability analysis of nonlinear compartmental models

Through use of the local state isomorphism theorem instead of the algebraic equivalence theorem of linear systems theory, the similarity transformation approach is extended to nonlinear models, resulting in finitely verifiable sufficient and necessary conditions for global and local identifiability. The approach requires testing of certain controllability and observability conditions, but in many practical examples these conditions prove very easy to verify. In principle the method also involves nonlinear state variable transformations, but in all of the examples presented in the paper the transformations turn out to be linear. The method is applied to an unidentifiable nonlinear model and a locally identifiable nonlinear model, and these are the first nonlinear models other than bilinear models where the reason for lack of global identifiability is nontrivial. The method is also applied to two models with Michaelis-Menten elimination kinetics, both of considerable importance in pharmacokinetics, and for both of which the complicated nature of the algebraic equations arising from the Taylor series approach has hitherto defeated attempts to establish identifiability results for specific input functions.     

Iterative image transformations for an automatic screening of cervical smears.

The new generation of image analysis systems permits the use of iterative image transformations. It is now possible to construct algorithms where the elementary steps are not arithmetic operations but image transformations. This will be illustrated by two examples. In the first, the absorption image of Feulgen Stained nuclei is processed by contrast algorithms in order to detect suspect cells. In the second, free lying cells are separated from overlapping cells and other artefacts by the use of skeletonization procedures.     

Colorimetry for the Stain Technologist. IV. Analysis of the Components of Color Difference

Total color differences have been calculated for various pairs of stained microscopic substrates. The latter include azure B/eosin stained blood cells and Papanicolsou stained cells from the uterine cervix. Both the CIE Luv and Lab color spaces have been used. Total color differences have been analyzed in terms of lightness, hue and chroma components. Various discrepancies have been noted among these components, especially the chroma difference, for the two spaces. It is concluded that current color-difference formulae are less than perfect, although they can provide much useful information.     

Colorimetry for the stain technologist. IV. Analysis of the components of color difference

Total color differences have been calculated for various pairs of stained microscopic substrates. The latter include azure B/eosin stained blood cells and Papanicolaou stained cells from the uterine cervix. Both the CIE L*u*v* and L*a*b* color spaces have been used. Total color differences have been analyzed in terms of lightness, hue and chroma components. Various discrepancies have been noted among these components, especially the chroma difference, for the two spaces. It is concluded that current color-difference formulae are less than perfect, although they can provide much useful information.     

Difluoro analogue of UCS15A triggers activation of exogenously expressed c-Src in HCT 116 human colorectal carcinoma cells

UCS 15A, an antibiotic produced by Streptomyces sp., has been reported to specifically disrupt SH3 domain-mediated interactions in eukaryotic cells. Interestingly, in the case of the non-receptor tyrosine kinase Src, UCS15A was effective in suppressing the SH3 domain-mediated intermolecular rather than intramolecular interactions, and thus prevented Src interactions with certain downstream effectors without affecting Src kinase activity. Here the synthesis of a novel difluoro analogue of UCS15A is described. The effects of this compound (8) on Src activity were tested in HCT 116 colorectal carcinoma cells engineered for inducible expression of c-Src. The presence of compound (8) resulted in the increased activity of the induced c-Src implicating that (8) acts as a c-Src activator in vivo. These observations are supported by computer modelling studies which suggest that the aldehyde group of (8) may covalently bind to a lysine residue in the SH2-kinase linker region situated in the proximity of the SH3 domain, which could promote a conformational change resulting in increased Src activity.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the "left endpoint" and "right endpoint" of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Dual-laser flow cytometry of single mammalian cells.

An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter signals from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter.     

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG-treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all-or-none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.     

Characterization of new bone morphogenetic protein (Bmp)‐2 regulatory alleles

Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial. A sequence within the 3'UTR of the Bmp2 mRNA termed the “ultra‐conserved sequence” (UCS) has been largely unchanged since fishes and mammals diverged. Cre‐lox mediated deletion of the UCS in a reporter transgene revealed that the UCS may repress Bmp2 in proepicardium, epicardium, and epicardium‐derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). The UCS also repressed the transgene in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra‐cardiac nerves, and neural ganglia. We used homologous recombination and conditional deletion to generate three new alleles in which the Bmp2 3'UTR was altered as follows: a UCS flanked by loxP sites with or without a neomycin resistance targeting vector, or a deleted UCS. Deletion of the UCS was associated with elevated Bmp2 mRNA and BMP signaling levels, reduced fitness, and embryonic malformations.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the “left endpoint” and “right endpoint” of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Recognition of cells in mitosis by flow cytofluormetry.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.     

Nonlinear optical properties of potential sensitive styryl dyes.

The nonlinear optical properties of dyes that alter their optical characteristics rapidly with membrane potential are described. The second harmonic signals from these dyes characterized in this paper are among the largest that have been detected to date. Structural conclusions are drawn from the second harmonic signals generated by the Langmuir Blodgett monolayers used in these measurements. Our results indicate that with appropriate instrumentation second harmonic signals could readily be detected from living cells stained with these dyes.     

Multidimensional scaling of color similarity in bees

A multiple choice experiment with free flying bees trained to a color signal is described which allows for multidimensional scaling of color similarity. The choice proportions are analysed by metric (Torgerson 1958) and non-metric (Kruskal 1964a, b) multidimensional scaling. The light reflected from the twelve color signals used differed in spectral composition, intensity, and the proportion of white light. Only two scales are necessary to reconstruct the experimental data. The interpretation of the scale values by Helmholtz-coordinates, derived from the chromaticity diagram for bees, shows that the main perceptual parameters are hue and saturation (or blue/greenness and UV/blue-greenness, respectively). Brightness is ignored by the bees in this choice situation. The total color difference is related to the differences on the two perceptual parameters by the city-block metric (Minkowski exponentp=1).