TECHNICAL NOTE |
Correspondence to: Rakesh K. Kumar, School of Pathology, University of New South Wales, Sydney, Australia 2052.
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Summary |
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Reliable double immunofluorescence labeling for confocal laser scanning microscopy requires good separation of the signals generated by the fluorochromes. We have successfully overcome the limitation of a single argon ion laser in achieving effective excitation of dyes with well-separated emission spectra by employing the novel sulfonated rhodamine fluorochromes designated Alexa 488 and Alexa 568. The more abundant antigen was visualized using the red-emitting Alexa 568, with amplification of the signal by a biotinylated bridging antibody and labeled streptavidin. This was combined with the green-emitting Alexa 488, which yielded brighter images than fluorescein but exhibited comparable photodegradation. With appropriate controls to ensure the absence of crosstalk between fluorescence channels, these dyes permitted unequivocal demonstration of co-localization. This combination of fluorochromes may also offer advantages for users of instruments equipped with alternative laser systems. (J Histochem Cytochem 47:12131217, 1999)
Key Words: immunohistochemistry, confocal laser scanning, microscopy, double labeling, fluorochromes
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Introduction |
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Confocal laser scanning microscopy (CLSM) permits accurate co-localization of fluorescent markers, because the thin optical section generated by the instrument eliminates the confounding effects of out-of-focus fluorescence (
For valid interpretation of the digital images generated by CLSM, reliable separation of the signals generated by the fluorochromes is critical. This is, in turn, dependent on the respective excitation/emission spectra and the choice of barrier filters. Overlap between the emission spectra of fluorescein and tetramethylrhodamine renders simultaneous excitation of probes labeled with these dyes unsatisfactory for CLSM (
An interesting solution to the problem of double immunofluorescence labeling for CLSM using a single argon ion laser was proposed by
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Materials and Methods |
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Antibodies and Labeled Probes
Mouse monoclonal anti-Type IV collagen, polyclonal rabbit antibodies to Ki-67 and to laminin, biotinylated polyclonal goat anti-mouse immunoglobulins, and swine anti-rabbit immunoglobulins labeled with fluorescein isothiocyanate (FITC) were purchased from Dako (Carpinteria, CA). Monoclonal anti-cytokeratins 4 and 13 were from Sigma (St Louis, MO). Goat anti-rabbit immunoglobulins labeled with Alexa 488 (excitationemission spectra similar to fluorescein) and streptavidin labeled with Alexa 568 (excitationemission spectra similar to lissaminerhodamine B) were from Molecular Probes (Eugene, OR).
Tissues and Immunostaining
Gingival tissues from individuals with advanced adult-type periodontitis had been collected as part of a previously described study (
Immunofluorescence Microscopy
Fluorescent staining of tissues was visualized using a Leitz Orthoplan microscope equipped with a xenon arc lamp, appropriate dichroics, and a x50, NA 1.0 water-immersion epifluorescence objective. Confocal laser scanning was performed with the Optiscan F900e personal confocal system (Optiscan; Melbourne, Australia), which uses an optical fiber both as the illumination source and the detection aperture. This system was equipped with a 50-mW argon ion laser, filters to attenuate the output of the laser to either 10% or 1% power, and filters allowing excitation with the 488-nm laser line, the 514-nm line, or a 50/50 split between the two wavelengths. Two channels were available for simultaneous data acquisition: Channel 1 (displayed as green) could include a 510550-nm bandpass filter and either a 515-nm or a 530-nm longpass filter, while Channel 2 (displayed as red) could include either a 550-nm or 590-nm longpass filter. Accumulated 16-scan digital images (512 x 512 x 16 bits per channel) were converted to bitmap files for printing.
Image Analysis
Measurement of pixel intensity was undertaken to compare the relative intensity of staining with Alexa 488 and fluorescein and to assess the photostablity of fluorochromes under the conditions of CLSM. For the green fluorochromes, nuclei of cycling cells stained with Ki-67 antibody and anti-rabbit immunoglobulins conjugated to either of these labels were examined. Series of nine consecutive 16-scan monochrome images were accumulated. A 128 x 128 pixel region of each image, containing a single optimally labeled nucleus with a visible nucleolus, was defined and mean pixel intensity for the region was determined using the Image/Histogram menu option in Adobe Photoshop software (Adobe Systems; San Jose, CA). To quantify relative intensity of staining with the two fluorochromes, the first 16-scan image of each series was analyzed. To quantify photodegradation, the procedure was repeated for all images in each series and the intensity expressed as a percentage of the initial value for that series. Photodegradation of Alexa 568 was similarly assessed by examining series of images of blood vessels stained for Type IV collagen with a streptavidinbiotin-amplified detection system. The software package GraphPad Prism (GraphPad Software; San Diego, CA) was used for data analysis and preparation of graphs.
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Results |
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Double Immunofluorescence
Initial experiments established that, using a biotinylated bridging antibody and streptavidin conjugated to Alexa 568, the red-emitting fluorochrome could be visualized by confocal microscopy when excited with the 488-nm line of an argon ion laser and detected using a 590-nm longpass filter. However, because excitation at this wavelength was inefficient, a high photomultiplier tube (PMT) gain was required to obtain a satisfactory image. As a result, when double immunofluorescence labeling was attempted, longer wavelength emissions from green fluorochromes such as Alexa 488 and FITC were amplified to unacceptably high levels, producing crosstalk from the green to the red channel.
Therefore, in subsequent experiments we used a 50/50 split of both 488-nm and 514-nm laser lines for excitation at 10% laser power. This yielded a satisfactory signal for both fluorochromes without inordinately high settings for PMT gain, using a 510550-nm bandpass plus a 530-nm longpass filter for detection of green emission in Channel 1 (the emission signal from 530550 nm was collected to exclude the 514-nm excitation line) and a 590-nm longpass filter for detection of red emission in Channel 2. As demonstrated in Figure 1A1C, with this arrangement there was no detectable crosstalk in either direction between the non-co-localizing signals from cytoplasmic staining of epithelial cells for keratins and staining of nuclei of cycling cells for Ki-67.
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Using appropriate settings for PMT gain, determined on the basis of non-co-localizing double staining for Ki-67 and cytokeratin, this combination of fluorochromes and filters also permitted demonstration of true co-localization. An example of co-localized matrix proteins in the thickened walls of blood vessels in advanced periodontitis is shown in Figure 1D1F, stained for Type IV collagen and for laminin.
Photostability of Fluorochromes
We found that using a secondary antibody labeled with Alexa 488 consistently yielded brighter images of nuclei stained with optimal concentrations of Ki-67 antibody than were obtained with fluorescein, but substantial fading was observed with both dyes under the experimental conditions employed (Figure 2A2F). These findings were confirmed by image analysis (Figure 3). In contrast, Alexa 568 exhibited no visible fading when used as a reporter label for blood vessels stained for Type IV collagen (Figure 2G2I) and no fading was demonstrable by image analysis (not shown).
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Discussion |
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Reliable double immunofluorescence labeling for confocal microscopy requires that the fluorochromes used exhibit good spectral separation. For confocal microscopes equipped with an argon ion laser with a limited output of laser lines, this is a difficult prerequisite to satisfy. A green-emitting dye such as fluorescein is efficiently excited by the 488-nm laser line, but a well-separated red-emitting fluorochrome such as Texas Red, which is widely used for conventional double immunofluorescence microscopy, is poorly excited using the available laser lines. In this study we have successfully implemented a solution originally proposed by
We used the novel fluorochromes designated Alexa 488 and Alexa 568 for these experiments. The Alexa dyes are sulfonated rhodamine derivatives, which are described by the manufacturer as brighter and more photostable than the traditional fluorochromes they replace. In our hands, these dyes proved very useful for visualizing double immunofluorescence using CLSM. Compared to fluorescein, brighter images were obtained using antibodies labeled with Alexa 488. However, whereas
For successful visualization of dual staining by CLSM using this approach, the more abundant antigen must be detected using the amplified reporter system and the red fluorochrome. As pointed out by
Photostability of fluorochromes is a major issue for CLSM, particularly for three-dimensional reconstruction of images. Green-emitting fluorochromes are a recognized problem. Alexa 488 has been demonstrated by the manufacturer to be more photostable than fluorescein for conventional immunofluorescence and was therefore investigated for CLSM. Unfortunately, in this study we found that photodegradation of the Alexa 488 dye under the conditions of CLSM was at least as marked as that observed with fluorescein. However, there was no apparent photodegradation of Alexa 568, indicating the potential of this fluorochrome for images using a single label for three-dimensional reconstruction. Indeed, the use of red-emitting dyes as the primary labels for immunofluorescence microscopy, including CLSM, has been strongly advocated (
The strategy for double immunofluorescence labeling that we have described for CLSM is not limited to systems equipped with an argon ion laser. Mixed gas argonkrypton lasers are able to optimally excite both of these Alexa dyes, which could be expected to yield increased green emission using Alexa 488 compared to fluorescein, because of the higher output, as well as increased red emission using Alexa 568 compared to Texas Red, because of more efficient excitation. Similar improvements could be anticipated using sequential excitation with an argon ion laser at 488 nm and a heliumneon laser at 543 nm in a dual laser system. In both cases, further amplification of the red signal during immunostaining is unlikely to be necessary.
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Acknowledgments |
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Supported by a grant from the Dental Board of New South Wales. CCC is a recipient of a Dental Postgraduate Scholarship from the National Health and Medical Research Council of Australia.
Received for publication December 1, 1998; accepted March 16, 1999.
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