TECHNICAL NOTE |
Correspondence to: Hans-Anton Lehr, University of Mainz, Medical Center, Inst. of Pathology, Langenbeckstr. 1, D-55101 Mainz, Germany..
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Summary |
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Simultaneous detection of two different antigens on paraffin-embedded and frozen tissues can be accomplished by double immunohistochemistry. However, many double chromogen systems suffer from signal overlap, precluding definite signal quantification. To separate and quantitatively analyze the different chromogens, we imported images into a Macintosh computer using a CCD camera attached to a diagnostic microscope and used Photoshop software for the recognition, selection, and separation of colors. We show here that Photoshop-based image analysis allows complete separation of chromogens not only on the basis of their RGB spectral characteristics, but also on the basis of information concerning saturation, hue, and luminosity intrinsic to the digitized images. We demonstrate that Photoshop-based image analysis provides superior results compared to color separation using bandpass filters. Quantification of the individual chromogens is then provided by Photoshop using the Histogram command, which supplies information on the luminosity (corresponding to gray levels of black-and-white images) and on the number of pixels as a measure of spatial distribution. (J Histochem Cytochem 47:119125, 1999)
Key Words: Photoshop, image analysis, immunohistochemistry, quantification
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Introduction |
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Double immunohistochemistry has been developed to allow the simultaneous detection of different antigens on cells contained on a tissue section. This technique is particularly important in situations where the immediate proximity of different cells or co-expression of different antigens on certain cells is being investigated. In addition, double immunohistochemistry is used when information on the spatial contribution of tissue elements (e.g., tumor cells vs stroma) is sought. Over the past few years the techniques of double immunohistochemistry have been refined considerably (
Photoshop, a program developed and constantly refined for the manipulation of digitized images, has become one of the essential tools in graphic design, desktop publishing, and advertising agencies. To allow manipulation of selected areas or features contained within the digitized image, Photoshop supplies sophisticated tools and commands for the recognition, selection, and separation of objects, shapes, gray levels, and colors. We show in this report that this latter feature of Photoshop can be applied to the differential selection and quantitative analysis of chromogens on digitized images taken from double immunostained microscope slides.
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Materials and Methods |
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Antibodies and Reagents
Mouse anti-CD68 (clone PG-M1), mouse smooth muscle anti--actin (clone 1A4), mouse anti-vimentin (clone V9), normal goat serum, alkaline phosphatase-conjugated streptavidin (strep/AP), biotinylated goat anti-mouse immunoglobulin (GAM/biotin), normal mouse serum, rabbit anti-fluorescein isothiocyanate (FITC), alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (GAR/AP), and New Fuchsin AP substrate system were from DAKO (Glostrup, Denmark). FITC-conjugated mouse anti-cytokeratin (clone CAM5.2) was from Becton Dickinson (San Jose, CA). Alkaline phosphatase-conjugated goat anti-mouse IgG3-specific (GAM-IgG3/AP), biotinylated goat anti-mouse IgG2a-specific (GAM-IgG2a/biotin), and ß-galactosidase-conjugated goat anti-rabbit immunoglobulin (GAR/GAL) were from Southern Biotechnology (Birmingham, AL). ß-Galactosidase-conjugated streptavidin (strep/GAL) and 5-bromo-3-chloro-2-indolyl-ß-D-galactopyaranoside (X-GAL) were from Boehringer Mannheim (Mannheim, Germany). Naphthol-ASMX phosphate, Fast Blue BB, ferrihexacyanide, and ferrohexacyanide were from Sigma (St Louis, MO). Antisera and antibody/enzyme conjugates were diluted in Tris-HCl (50 mM, pH 7.8)-buffered saline (TBS) + 1% bovine serum albumin (BSA). TBS washings were performed between all steps (three times for 2 min) and all incubations were performed at room temperature (RT) unless otherwise stated.
Immunohistochemical Technique
Human carotid artery segments with atherosclerotic lesions were fixed in phosphate-buffered formaldehyde for 2472 hr and then embedded in paraffin. Five-µm-thick sections were mounted on organosilane-coated slides and dried overnight at 37C. Sections were deparaffinized in xylene, rehydrated in graded alcohols, and washed with tapwater. Then the sections were treated for antigen retrieval using citrate (10 mM, pH 6.0) in a household microwave oven (
Breast carcinoma specimens were snap frozen in isopentane-cooled liquid nitrogen and stored at -80C. Five-µm sections were cut, dried overnight under a fan at room temperature, and either used right away or stored dry in a closed box at -80C. Cryostat sections were acetone-fixed (10 min, 4C), briefly air-dried, and extra fixed to preserve a better tissue morphology with Zamboni's fluid (picric acid/paraformaldehyde in phosphate buffer, pH 7.4) for exactly 2 min, washed with TBS (three times for 2 min), and then covered with normal goat serum (1:10, 15 min). Double labeling was based on either two primary antibodies from different IgG subclasses (CD68/-actin; Figure 1), or one unlabeled primary antibody and one FITC-conjugated antibody (vimentin/cytokeratin; Figure 2) (
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From the available color combinations for double staining, we selected one with a superior color contrast: turquoise/red, as obtained with ß-galactosidase and alkaline phosphatase as tracer enzymes, and X-Gal and New Fuchsin as chromogens, respectively (-actin double staining applied on paraffin sections from the carotid artery (Figure 1): mouse anti-CD68, PG-M1 (IgG3) (1:50) in a cocktail with mouse anti-
-actin, 1A4 (IgG2a) (1:100, 60 min); GAM-IgG3/AP (1:20) in a cocktail with GAM-IgG2a/biotin (1:100, 30 min); strep/GAL (1:40, 30 min). The following subsequent incubations were performed for the red/turquoise vimentin/cytokeratin double staining applied on paraffin sections from breast carcinoma (Figure 2): anti-vimentin (1:200, 60 min); GAM/biotin (1:200, 30 min); cocktail of strep/AP (1:100) and normal mouse serum (1:10, 30 min); FITC-conjugated anti-cytokeratin (1:20, 60 min); rabbit anti-FITC (1:1000, 15 min); and finally GAR/GAL (1:10, 60 min).
Double staining specific controls consisted of replacing either one of both primary antibodies by nonimmune reagents of the same species or IgG subclass, keeping the protein concentration similar to the primary antibodies. After all antibody detection steps the enzymatic activities were visualized. For the red/turquoise color combination, first ß-galactosidase activity was developed with X-GAL and ferro-ferricyanide (
Image Analysis
Images are imported into the S-VHS port of a personal computer (G3 Power Macintosh with inbuilt graphic capture board) using a one-chip CCD red-green-blue (RGB) color video camera (JVC TK-C1381 camera; Tokyo, Japan) and a standard diagnostic microscope (Dialux 22; Leitz, Wetzlar, Germany) equipped with a halogen light source connected to a stabilized, adjustable power supply (12 V, 100 W). Images are opened in Photoshop (version 4; Adobe Systems, San Jose, CA) and stored in a Photoshop or a PICT file format on the hard drive or on an external data storage device (ZIP drive; Iomega, Roy, Utah).
The technique of selection of similar features on a digitized immunohistochemical image has previously been described in detail (
Once the different chromogens are selected, quantification is accomplished using the Histogram command in the Image menu. This display is rarely if ever used by graphic designers but rather serves as an internal measurement of tonal distribution as the basis for automated image manipulation (map commands). When Histogram is selected, a display appears on the screen depicting the gray levels (black/white) or the luminosity (color) of all pixels within the selected area, including median and standard deviation. Furthermore, this display shows the number of pixels that are covered by the selected area. Because the number of pixels reflects a surface area on the image, important spatial information can be obtained for the specific chromogen (and hence the cells expressing a certain antigen) and can be expressed as percentage of the entire image or in µm2.
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Results |
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We demonstrate in this study that complete chromogen separation is possible using Photoshop-based image analysis. In contrast to color separation by the use of bandpass filters, which only incompletely eliminated the respective chromogen on the image (Figure 1AD), Photoshop-based image analysis was found to completely separate the two chromogens (Figure 1EG). When applied to breast cancer tissue composed of epithelial cells (cytokeratin antibody, New Fuchsin, red chromogen) and stroma (vimentin antibody, ß-Gal, turquoise chromogen), we found that color separation was complete. The surface areas of epithelial cells and stroma, assessed as numbers of pixels covered by the respective chromogens using the Histogram tool, added up to almost 100% of the entire tumor field (70.2 + 30.3%; Figure 2). A similar result was obtained when chromogen separation and quantification were performed on a random high-magnification section of the tumor (Figure 2, insets), demonstrating that Photoshop-based image analysis allowed complete chromogen separation irrespective of the image magnification.
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Discussion |
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The present study demonstrates that complete chromogen separation is possible on digitized multispectral images using Photoshop-based image analysis. This software program makes use of the integral information on color hue, saturation, and luminosity of every individual pixel in a digitized image. To better visualize and hence to control the efficacy of color separation, manipulations of saturation and/or hue can be performed on the different colors contained in the image. This is best demonstrated in Figure 1B and Figure 1E and in Figure 2A and Figure 2B, in which the luminosity of the turquoise chromogen is enhanced and the red chromogen is turned into a bright pink color. Although this manipulation does not affect the ability of the software to separate the colors, it does help to control the effective separation of the two chromogens on the computer monitor.
Over the past decade, color separation on digitized images of immunohistochemical slides has been performed successfully using diverse custom-made image analysis programs such as IBAS 2000 (
In further developments of the described techniques, RBG thresholding was improved considerably by integrating into the thresholding algorithm information on hue, luminosity, and saturation (Gato et al. 1992;
As a practical example, we applied Photoshop-based color separation to the selective quantification of breast carcinoma tissue (Figure 2) covered by epithelial tumor cells (cytokeratin antibody, New Fuchsin, red chromogen) or by stroma (vimentin antibody, ß-Gal, turquoise chromogen). We found that, in addition to complete chromogen separation, the calculated surface area of epithelium and stroma added up to almost 100% of the selected tumor field. Of particular interest is the fact that color separation is effectively performed at both low (Figure 2) and high magnifications (Figure 2, insets). In contrast to RGB thresholding techniques, which demonstrate improved separation capacities at higher magnifications (
There are only a few reports in the literature in which Photoshop has been applied to biomedical research. Several authors have used Photoshop merely as a way of importing scanned images into their computers for later analysis by other programs (
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Acknowledgments |
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We are indebted to Drs Frans Nauwelaers and Peter Oud for the idea of applying a narrow bandpass filter to the digitized images.
Received for publication August 6, 1998; accepted August 25, 1998.
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