Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

A selective stain for mitotic figures, particularly in the developing brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

Quantification of histochemical staining by color deconvolution

OBJECTIVE: To develop a flexible method of separation and quantification of immunohistochemical staining by means of color image analysis. STUDY DESIGN: An algorithm was developed to deconvolve the color information acquired with red-green-blue (RGB) cameras and to calculate the contribution of each of the applied stains based on stain-specific RGB absorption. The algorithm was tested using different combinations of diaminobenzidine, hematoxylin and eosin at different staining levels. RESULTS: Quantification of the different stains was not significantly influenced by the combination of multiple stains in a single sample. The color deconvolution algorithm resulted in comparable quantification independent of the stain combinations as long as the histochemical procedures did not influence the amount of stain in the sample due to bleaching because of stain solubility and saturation of staining was prevented. CONCLUSION: This image analysis algorithm provides a robust and flexible method for objective immunohistochemical analysis of samples stained with up to three different stains using a laboratory microscope, standard RGB camera setup and the public domain program NIH Image.     

Automatic leukocyte classification using cytochemically stained smears.

A leukocyte classification algorithm suitable for automated differential counting has been developed for blood smears stained with a new three-component cytochemical stain which has relatively narrow absorption bands centered at 460, 540 and 640 nm, respectively. The classification procedure is the result of a pattern recognition experiment using a sample of 223 leukocytes distributed evenly over the five normal cell types. The basic data for each cell were three digital microscopic images obtained with narrow band illumination at the above central wavelengths using a TV-digitizer system interfaced to a PDP-15 computer. The classification algorithm involves a sequential decision procedure utilizing five pattern features computed from the intensity histograms of the green and blue digital images. Thus the number of arithmetic operations and the number of computer memory words necessary to perform the classification into one of the five normal white blood cell types are both proportional to n where n is the number of gray levels into which the intensity scale is divided. In this experiment, n equals 256. Comparison of our results with work of others on smears prepared with Romanowski-type stains indicates that such narrow-band, spectrally well separated cytochemical multiple stains can permit the use of algorithms which are approximately ten times faster.     

Simplified heavy metal staining techniques demonstrated with Fast Plant leaf tissue

Fast Plant(Brassica rapa,Cruciferae)leaf tissuefixed in glutaraldehyde-acrolein and post-fixed in os-mium,was examined for response to several easily-prepared heavy metal stains.Lead and uranium,separately and in combination,gave typical resultsacross the spectrum of cell organellets.As a single stainfollowing osmium,bismuth produced images seeminglyequivalent to lead and uranium.Phosphotungstic acidproduced very good membrane delineation but produceda washed-out background image similar to that from leadstaining.Carbohydrate compounds were especiallyresponsive to ruthenium;the cytoplasm and the matrixof all organelles were also stained very well.Theprocedures were no more demanding than traditionalstaining methods and may be easily used in research andteaching.Fast Plant materials are a reliable,quick andeasy source of living material.     

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.     

Dual wavelength scanning cytophotometry (Bicoscan).

A computer program has been developed for stage-scanning cytophotometry of double-stained microscopical specimens. The program permits the simultaneous measurement of absorbance values at two wavelengths in each measuring spot. To account for overlap in the absorbance spectra of the two stained endproducts, the program incorporates correction of the measured data to compensate for the contribution of each chromophore to the absorbance measured for the other. The program will compute the corrected local absorbance values at specified wavelengths for each chromophore at each measuring spot and integrate these values over the total object to give separate totals for each stain. It is also possible to have integrated the absorbance values of one chromophore for all those measuring spots where the local corrected absorbance value of the other chromophore exceeds a preset minimum value. When this other chromophore is a nuclear DNA stain, it is possible to obtain an approximate measure of the content of any compound in the nuclear area which can be stained with a chromophore having an absorbance spectrum different from the DNA stain. The validity of the program was investigated on model preparations consisting of two (differently) coloured films of which the absorbance values could be measured either individually or in combination, by partially overlaying one film on the other. The program's potential has been demonstrated by using the combination of either Naphthol Yellow S or dinitrofluorobenzene as protein stains with the Feulgen-pararosaniline(SO2) procedure for DNA in chicken erythrocytes and rat liver cells.     

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.     

A Microspectrophotometric Analysis of Staining with Carmine, Orcein and Carmine-Orcein

Microspectrophotometric measurements of carmine, orcein and carmine-orcein were made in solutions, in air-dried films and in stained adult and embryonic tissues of the domestic chicken. For individual stains only minor differences were found between dried dye and stained tissue. The absorption curve for carmine in solution showed a single peak at 490 mμ but was bimodal at about 530 and 570 mμ in dry films and stained tissue. Orcein showed a single broad peak at 510 mμ in solution; in dry films and stained tissue a broadening of the absorption curve in the red wavelengths was observed. The dye mixture carmine-orcein in solution showed a single peak at 500 mμ, but in tissue the spectral absorptions closely resembled carmine. With alum-like carmine, spectral changes due to the addition of iron were not detected. The results indicate that nuclear staining with carmine-orcein is due mainly to the carmine component of the mixture. Interpretation of spectral shifts indicates that acew-carmine is a metachromatic stain while aceto-orcein is mainly an ortho-chromatic stain, although some metachromasy is evident.     

Structured Illumination Microscopy and a Quantitative Image Analysis for the Detection of Positive Margins in a Pre-Clinical Genetically Engineered Mouse Model of Sarcoma

Intraoperative assessment of surgical margins is critical to ensuring residual tumor does not remain in a patient. Previously, we developed a fluorescence structured illumination microscope (SIM) system with a single-shot field of view (FOV) of 2.1×1.6 mm (3.4 mm²) and sub-cellular resolution (4.4 μm). The goal of this study was to test the utility of this technology for the detection of residual disease in a genetically engineered mouse model of sarcoma. Primary soft tissue sarcomas were generated in the hindlimb and after the tumor was surgically removed, the relevant margin was stained with acridine orange (AO), a vital stain that brightly stains cell nuclei and fibrous tissues. The tissues were imaged with the SIM system with the primary goal of visualizing fluorescent features from tumor nuclei. Given the heterogeneity of the background tissue (presence of adipose tissue and muscle), an algorithm known as maximally stable extremal regions (MSER) was optimized and applied to the images to specifically segment nuclear features. A logistic regression model was used to classify a tissue site as positive or negative by calculating area fraction and shape of the segmented features that were present and the resulting receiver operator curve (ROC) was generated by varying the probability threshold. Based on the ROC curves, the model was able to classify tumor and normal tissue with 77% sensitivity and 81% specificity (Youden’s index). For an unbiased measure of the model performance, it was applied to a separate validation dataset that resulted in 73% sensitivity and 80% specificity. When this approach was applied to representative whole margins, for a tumor probability threshold of 50%, only 1.2% of all regions from the negative margin exceeded this threshold, while over 14.8% of all regions from the positive margin exceeded this threshold.     

Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei.

BACKGROUND: Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens. METHODS: Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data. RESULTS: The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample. CONCLUSIONS: The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.     

Quantitative peroxidase-antiperoxidase complex-substrate mass determination in tissue sections by a dual wavelength method.

The aim of this study was to develop a method of quantitating prostate-specific acid phosphatase (PSAP) in histologic sections of prostate tumor tissue labeled with the peroxidase-antiperoxidase (PAP) complex technique using diaminobenzidine (DAB) as a substrate. Studies of PAP-DAB- and hematoxylin-stained prostate tissue sections were performed with a black-and-white, computerized microscope image system. The mass of brown reaction product generated in PAP-DAB staining was the indicator of PSAP intensity. The mass of brown reaction product was determined by using a dual wavelength method in which two 10-nm bandpass filters, peaked at 450 and 510 nm in wavelength, were used. The wavelength-dependent ratio of mass absorptivity of PAP-DAB stain (brown product) and that of hematoxylin (blue product) were estimated at wavelengths of 450 and 510 nm by using slides stained with only PAP-DAB or hematoxylin. The accuracy of the mass measurements, investigated by relating the measurement to the true mass of the brown PAP-DAB product, is reported. There was no significant difference between the measurements at magnifications of 10x, 20x, 40x or 60x in the reproducibility investigation. The PSAP stain intensity was quantitatively determined by the difference of the PAP-DAB stain mass per pixel between the tumor and normal cell region. The relationship between the objective measurement and the conventional subjective grades is presented.     

Proliferation and apoptosis measurements by color image analysis based on differential absorption.

Cell proliferation and apoptosis indices are important indicators for the prognosis and treatment of a variety of cancers. A method is described using differential absorption color image analysis to measure proliferation and apoptosis in tumor sections using BrdU (5' bromodeoxyuridine) incorporation and immunohistochemistry and terminal deoxytransferase nick end-labeling (TUNEL). Nuclei were labeled with streptavidin-peroxidase-diaminobenzidine (DAB) secondary detection. The differential absorption method uses a computer-controlled microscope equipped with a tunable filter and digital camera to take advantage of the spectral differences of stained objects of interest. Images collected at defined wavelengths are divided and scaled to form ratio images in which the hematoxylin- or DAB-stained nuclei have intensity ranges far above those of surrounding structures. Using brightness thresholding followed by selection based on nuclear size and shape parameters, binary images were formed of the BrdU/apoptotic-positive tumor and all the tumor nuclei for subsequent counting and calculations of proliferation and apoptotic indices.