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.     

Hair color measurement and variation

Pigmentation of hair in humans has been investigated by medical scientists, anthropologists and, more recently, by forensic scientists. In every investigation, hair color must first be defined by the researchers. Subjective color assessment inhibits the reproducibility of experiments and the direct comparison of results. The aim of this study was to objectively measure human hair color and examine the variation found in a population with European ancestry, using the CIE L*a*b* color space. Observer-perceived hair colors were compared with self-reported hair colors and the color as measured by reflective spectrophotometry of 132 subjects of European ancestry. The presented data show that self-reported hair colors and observer-reported colors are similar; however, these categories are not necessarily the best way to categorize hair color for quantitative research. Using a two-step cluster analysis, hair color can be divided into categories or clusters based on spectrophotometric measurements in the CIE L*a*b* color space and these clusters can be well discriminated from each other. This separation is primarily based on the b* (yellow) color component and the clusters show agreement to observer-reported colors. This study illustrates the possibilities for and necessity of objectively defining the hair color phenotype for various downstream applications.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Accuracy of color prediction of anthraquinone dyes in methanol solution estimated from first principle quantum chemistry computations

The electronic spectrum of four different anthraquinones (1,2-dihydroxyanthraquinone, 1-aminoanthraquinone, 2-aminoanthraquinone and 1-amino-2-methylanthraquinone) in methanol solution was measured and used as reference data for theoretical color prediction. The visible part of the spectrum was modeled according to TD-DFT framework with a broad range of DFT functionals. The convoluted theoretical spectra were validated against experimental data by a direct color comparison in terms of CIE XYZ and CIE Lab tristimulus model color. It was found, that the 6-31G** basis set provides the most accurate color prediction and there is no need to extend the basis set since it does not improve the prediction of color. Although different functionals were found to give the most accurate color prediction for different anthraquinones, it is possible to apply the same DFT approach for the whole set of analyzed dyes. Especially three functionals seem to be valuable, namely mPW1LYP, B1LYP and PBE0 due to very similar spectra predictions. The major source of discrepancies between theoretical and experimental spectra comes from L values, representing the lightness, and the a parameter, depicting the position on green→magenta axis. Fortunately, the agreement between computed and observed blue→yellow axis (parameter b) is very precise in the case of studied anthraquinone dyes in methanol solution. Despite discussed shortcomings, color prediction from first principle quantum chemistry computations can lead to quite satisfactory results, expressed in terms of color space parameters.     

Quantitative and qualitative analysis of stain color using RGB computer color specification

OBJECTIVE: To establish standardized Papanicolaou stain for cytology using RGB color specification. This new method was formerly used in DTP software application for computer color specification. STUDY DESIGN: RGB color specification was taken from a color film, optical constituents of which were made into computer software. Cell samples used in this study were from 100 sputum specimens stained with Papanicolaou stain. We analyzed the color tone of the cytoplasm of squamous cells in the smear. RESULTS: The R and B value of eosinophilic cells were demonstrated statistically by different values between normal, borderline atypical and malignant squamous cells. G and B values of light green-philic cells demonstrated a statistical difference between normal, borderline atypical and malignant squamous cells. No significant differences were found in RGB value between normal, borderline atypical and malignant squamous orangeophilic cells. CONCLUSION: Using our own method of analyzing Papanicolaou-stained sputum, a new quantitative and qualitative analysis of stain color for standardized Papanicolaou stain was introduced.     

Technical note: quantitative measures of iris color using high resolution photographs

Our understanding of the genetic architecture of iris color is still limited. This is partly related to difficulties associated with obtaining quantitative measurements of eye color. Here we introduce a new automated method for measuring iris color using high resolution photographs. This method extracts color measurements in the CIE 1976 L*a*b* (CIELAB) color space from a 256 by 256 pixel square sampled from the 9:00 meridian of the iris. Color is defined across three dimensions: L* (the lightness coordinate), a* (the red-green coordinate), and b* (the blue-yellow coordinate). We applied this method to a sample of individuals of diverse ancestry (East Asian, European and South Asian) that was genotyped for the HERC2 rs12913832 polymorphism, which is strongly associated with blue eye color. We identified substantial variation in the CIELAB color space, not only in the European sample, but also in the East Asian and South Asian samples. As expected, rs12913832 was significantly associated with quantitative iris color measurements in subjects of European ancestry. However, this SNP was also strongly associated with iris color in the South Asian sample, although there were no participants with blue irides in this sample. The usefulness of this method is not restricted only to the study of iris pigmentation. High-resolution pictures of the iris will also make it possible to study the genetic variation involved in iris textural patterns, which show substantial heritability in human populations.     

Discriminating between damaged and intact cells in fixed flow cytometric samples

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.     

Photography in limnology: documentation of lake color using a CCD camera

During a survey of Norwegian lakes, photographic records were made of lake color as reflected by a white Secchi disk positioned at half of the depth of extinction. The pictorial distinction between different lakes is documented in this article. The pictures recorded can readily be transferred to a color model [e.g., Commission Internationale de L’Éclairage (CIE)-Lab]; from there, numerical color parameter values such as hue (h*, the quality of color) and chroma (C*, the intensity of color) may be assigned to each record. There are, however, limitations and obstacles connected with the transformation of color values recorded by a CCD camera (or film digitizers) to absolute numerical values comparable between different cameras and systems. A single CCD camera may be useful for documenting lake color, but there are technical limitations restricting its use as a scientific instrument for quantitative purposes.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

色彩间接效应：现象学和时间现论

色彩间接效应即色彩通过社觉产生的生理和心理效应。本文从中西结合医学的角度研究了ＤＣＩＥ,提出了色－植物神经模型,并建立了相应的时间理论。     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

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.     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa

OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.     

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.     

Two staining methods for selectively detecting isomaltase and maltase activity in electrophoresis gels.

Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.     

Cytophotometry of breast carcinoma. Acridine-orange DNA microfluorimetry with Giemsa counterstain

Investigations have suggested that a correlation exists between DNA ploidy levels and prognosis in human breast carcinoma. Nuclear DNA content can be studied by flow cytometry or cytophotometric analysis. While both methods yield comparable results for DNA distribution, cytophotometry has the advantage of permitting both quantitative cell measurements and cytomorphologic identification of tumor cells. Microfluorimetric analysis of nuclear DNA content was carried out on acridine-orange-stained imprint smears of malignant breast tumors, with the DNA values plotted as a histogram distribution. Quantitative fluorescence measurements of breast carcinoma cells using the acridine-orange stain appeared to be a fairly rapid and simple method for DNA determination as compared to Feulgen DNA analysis. Following cytometric measurements, imprint smears were counterstained by the Giemsa stain and examined by cytomorphologic criteria. With the Giemsa counterstain, the same cytologic preparation could be studied both by quantitative cell measurements and by conventional cytomorphologic criteria. Results are illustrated, and possible implications of the use of this method in the study of tumor behavior and the diagnosis by cytologic methods are discussed.     

The basis for colored silver-protein complex formation in stained polyacrylamide gels

Using a modified silver stain of Merril et al. [(1981) Science 211, 1437-1438] for staining polypeptides in sodium dodecyl sulfate-polyacrylamide gels, protein bands reproducibly stain different shades of blue, yellow, red, and gray. The procedure is highly temperature dependent, with optimal color formation at 42 degrees C. The procedure may be completed within 2 h. Color formation is due to silver ion complexes with charged amino acid side chains. The color of the silver-protein complex can be predicted if the amino acid sequence is known, although some exceptions are discussed. This provides another dimension to the characterization of proteins by gel electrophoresis.     

Red Food Coloring Stain: New,Safer Procedures for Staining Nematodes in Roots and Egg Masses on Root Surfaces

Acid fuchsin and phloxine B are commonly used to stain plant-parasitic nematodes in roots and egg masses on root surfaces, respectively. Both stains can be harmful to both the user and the environment and require costly waste disposal procedures. We developed safer methods to replace both stains using McCormick Schilling red food color. Eggs, juveniles, and adults of Meloidogyne incognita stained in roots with red food color were equally as visible as those stained with acid fuchsin. Egg masses stained with red food color appeared as bright-red spheres on the root surfaces and were highly visible even without magnification. Replacement of acid fuchsin and phloxine B with red food color for staining nematodes is safer for the user and the environment, and eliminates costly waste disposal of used stain solutions.