Jan Michels, Department of Functional Morphology and Biomechanics, Institute of Zoology, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, D-24098 Kiel, Germany. Tel: +49 431 880-4511; fax: +49 431 880-1389; e-mail: firstname.lastname@example.org
In this study, the potential of the common dye Congo red as a fluorescence marker for chitin in the exoskeleton of small crustaceans and collagen in the polychaete cuticle was tested. The Congo red staining turned out to be rather efficient and yielded intensively fluorescing structures, which made a very detailed visualization by confocal laser scanning microscopy possible. The excellent results are comparable to those described for the utilization of other efficient fluorescence dyes and intense autofluorescence. The application of Congo red is easy, the fluorescence of this dye is very stable, and the excitation maximum of the structures stained with Congo red is in a range, which is covered by the lasers of most of the confocal laser scanning microscopes. These advantageous properties make the fluorescence staining by Congo red a method of choice for the detailed visualization of the external morphology of small crustaceans and polychaetes.
In recent years, several studies have demonstrated the potential of cuticular autofluorescence and certain fluorescence dyes for the detailed visualization of insect and crustacean morphology, mainly in combination with confocal laser scanning microscopy (CLSM) (Galassi et al., 1998; Carotenuto, 1999; Zill et al., 2000; Zupo & Buttino, 2001; Buttino et al., 2003; Klaus et al., 2003; Moussian et al., 2005; Schawaroch et al., 2005; Tonning et al., 2005; Klaus & Schawaroch, 2006; Michels, 2007; Maruzzo et al., 2009). In the framework of taxonomic studies on copepods, we tested some of the described methods and found that in a considerable number of species the autofluorescence of the exoskeleton is not intensive enough to visualize the external morphology in a satisfying way. Maruzzo et al. (2009) digested copepod nauplii using KOH and stained the remaining exoskeleton with an unspecific dye. This method is very effective, however, often type specimens, which must not be damaged, have to be investigated in the context of taxonomical studies. Accordingly, this digestion method cannot be used. Furthermore, many of the yet applied fluorescence dyes have disadvantages with regard to their applicability as they do not exclusively stain specific exoskeleton components such as chitin (e.g. DiI), or bleach rather fast (e.g. FITC-conjugated chitin-binding probe) so that precise CLSM scans of relatively thick samples are not feasible. Other potential dyes, as for example Calcofluor White, which specifically stains chitin (e.g. Roncero & Durán, 1985) and thus may be very appropriate to visualize insect and crustacean exoskeletons, have excitation maxima in the UV range. As not all confocal laser scanning microscopes are equipped with UV lasers, such dyes can be applied only by a limited number of scientists. As a consequence of these disadvantages, we searched for a fluorescent dye that stains chitin, is easy and effective to apply and does not bleach too fast so that complete crustaceans and other small organisms can be scanned and visualized in detail by CLSM. In this context, we came upon the fluorescent dye Congo red, which had been shown to effectively stain chitin in fungi and arthropods (Schmidt, 1954, and citations therein; Slifkin & Cumbie, 1988; Matsuoka et al., 1995; Tonning et al., 2005). The main goal of the present study was to assess the efficiency and intensity of Congo red staining of chitin in the exoskeleton of small crustaceans in order to visualize the external morphology of the respective specimens. For this purpose, different crustaceans from the taxa Copepoda, Phyllopoda and Eumalacostraca were used. In addition, as Congo red is not chitin-specific and also stains, amongst other materials, collagen (Elghetany et al., 1989, and citations therein; Bély & Makovitzky, 2006, and citations therein), we decided to test Congo red as a fluorescence marker for the polychaete cuticle, which contains no chitin but considerable amounts of collagen (e.g. Spearman, 1973; Nielsen, 2001; Purschke, 2002; Hausen 2005). Small marine polychaetes collected in the northern North Sea were used as test organisms. The evaluation of the potential of Congo red for a detailed visualization of the external morphology of small crustaceans and polychaetes was performed using CLSM.
Material and methods
Fixation and Congo red staining. Before the preparation all animals were fixed and stored in formalin (4% formaldehyde/water solution) or ethanol (98%). The staining solution was prepared by dissolving Congo red (Sigma-Aldrich, Steinheim, Germany) in distilled water (1.5 mg per millilitre of distilled water). In order to remove particles, some millilitres of the solution were filtered through a syringe filter (Filtropur S 0.2 (Sarstedt AG & Co., Nümbrecht, Germany)) directly into the staining glass vial just before each staining process. The specimens were thoroughly washed in distilled water, added to the Congo red solution by thin glass pipettes and stained at room temperature for 24 h. Afterwards, they were transferred into distilled water by thin glass pipettes, left there for 5 min and then washed thoroughly in distilled water several times until no solute Congo red was present anymore.
Mounting. The animals were mounted in self-made microvials on objective slides (Fig. 1). For the preparation of these microvials, transparent self-adhesive reinforcement rings (normally used for office work) were glued to the slide as described by Kihara & Falavigna da Rocha (2009). Two to six reinforcement rings were stacked, depending on the size of the animals. This was necessary to avoid contact between the specimen and the cover slip, which may cause reflections or interferences in the detected light. Inside these microvials, the specimens were mounted in glycerine or, if they tended to shrink in glycerine, a mixture of glycerine and water. The microvials were covered with 0.17-mm-thick cover slips (refractive index = 1.5255), holding onto the vials only by adhesion and thus allowing a change of the orientation of the specimens by moving the cover slip gently.
Confocal laser scanning microscopy imaging. All specimens were viewed on a Leica TCS SP5 (Leica Microsystems GmbH, Wetzlar, Germany) equipped with an upright microscope (Leica DM5000 B) and three visible light lasers (Ar 100 mW 458 nm, 476 nm, 488 nm and 514 nm; DPSS 10 mW 561 nm; HeNe 10 mW 633 nm). Dependent on the size of the animals different chromatically corrected lenses were used (see Table 1). When testing all available excitation wavelengths, we found that the optimal fluorescence signal of Congo red was stimulated by the 561 nm laser line. Accordingly, this wavelength was used for our evaluation of Congo red. The laser power was set to 75%. It is relatively unusual to use such a high laser power, however, our intention was to test the stability and the bleaching characteristics of the Congo red staining. The emitted fluorescent light was detected in the range from 570 nm to 670 nm. These settings were applied for all CLSM images created in the present study. For each preparation amplitude offset and detector gain were manually adjusted and image stacks were collected as described by Michels (2007). The imaging parameters of all CLSM image stacks are given in Table 1. Based on the image stacks, maximum intensity projections were created with the Leica LAS software (Leica Microsystems GmbH, Wetzlar, Germany). The final images were adjusted for contrast and brightness using the software Nikon Capture NX 2 (Nikon Corporation, Tokyo, Japan) and Adobe Photoshop CS4 (Adobe Systems, San José, California, U.S.A.).
Table 1. Overview of the embedding media, microscope lenses, immersions, types of fluorescence and CLSM settings used for the visualization of the different specimens. CLSM settings, which are given just once in the middle row of the table, were applied for all specimens.
To demonstrate the efficiency and intensity of the Congo red staining, we decided to visualize specimens of the copepod species Paramphiascella sp., whose exoskeleton has only weak autofluorescence, with different methods: (1) one female and one male were stained with Congo red and visualized using optimized CLSM settings; (2) one unstained female was visualized using cuticular autofluorescence and the identical CLSM settings used for the stained female mentioned in (1); (3) the same unstained female as in (2) was also visualized using cuticular autofluorescence and optimized CLSM settings; (4) in addition, the same female as in (2) and (3) was subsequently stained with Congo red and visualized using optimized CLSM settings. (2)–(4) were performed in order to clearly illustrate the difference between the autofluorescence of the exoskeleton and the fluorescence of the Congo red stained exoskeleton.
Results and discussion
Congo red proved to intensively stain chitin in the exoskeleton of small crustaceans and collagen in the cuticle of polychaetes. When applying the described staining method in combination with CLSM, we obtained maximum intensity projections that show the external morphology of the investigated organisms in great detail (Figs 2(a), (b), (e) and 3). The Congo red stained materials have a strong fluorescence, and even tiny structures such as setae and thin, pointed parts of the mouthparts and swimming legs (e.g. Figs 2(a), (b), (e) and 3(a–c)) as well as chaetae of polychaetes (Fig. 3(f)) and surface microstructures of, e.g., the carapace of daphnids (Fig. 3(c)) were efficiently stained and could be visualized successfully. This is in accordance with earlier observations showing that Congo red stains the chitin-containing parts of the exoskeleton very intensively and thus enables the analysis of even the smallest chitinous structures (Schmidt, 1954). The ability of Congo red to efficiently stain cellulose and chitin (polysaccharides), as well as amyloids, in plants and animals was already known several decades ago (Schmidt, 1954, and citations therein), and since then this dye has become frequently used in histological studies. However, in the framework of these observations, the properties of Congo red as a fluorescence marker for the exoskeleton were not investigated.
The tests of our study revealed that Congo red is rather easy to apply and has the great advantage of being a very stable fluorescence dye. After exposing the stained preparations for scan times of 1.5–2.5 h to laser light, which was generated with a laser power of 75%, the bleaching effects were negligible. And when applying a laser power of 20–35%, we even did not observe mentionable bleaching after scan times of about 6 h.
Our results show that the Congo red staining is clearly restricted to the exoskeleton and the cuticle, whereas internal tissues are not stained. This is illustrated using the example of the stained exoskeletons of two female Paramphiascella sp. (Fig. 4). As proteins are not stained by Congo red (Schmidt, 1954), the method described here can not only be used to analyze the external morphology of small crustaceans and polychaetes in great detail but also to selectively visualize exoskeleton components, which are internal, within sections.
The functional principle of the chitin staining is due to an intercalation of Congo red between the chitin chains (Cohen, 1993, and citations therein). The diminutive, dichroic Congo red particles are all orientated in the same direction along the chitin chains resulting in a summation of their optical properties (Schmidt, 1954, and citations therein). In this context, our results show that different types of chitin are stained by Congo red: α-chitin, which is present in the exoskeleton of crustaceans (e.g. Ifuku et al., 2009) (Figs 2(a), (b), (e) and 3(a–e)), and β-chitin, which is present in the chaetae of polychaetes (e.g. Purschke, 2002; Fan et al., 2008, and citations therein) (Fig. 3(f)).
When visualizing important samples such as type specimens of museum collections, one disadvantage of the Congo red staining might be the red colouration of the specimens. However, we observed that this colouration is not permanent and fades considerably when the stained specimens are rinsed with ethanol. After having been stored in ethanol for some days, these specimens exhibit just a slight red shimmer, and after additional rinsing with ethanol and some further days of storage in ethanol there is nearly no staining left. We assume that this is due to the Congo red particles, which intercalate between the chitin chains and collagen fibres, dissolving in ethanol after some time. Accordingly, Congo red staining should not cause any concerns from responsible persons such as museum collection managers.
In overviews and descriptions of fluorescence dyes 490 nm is given as excitation maximum of Congo red (pers. comm. Gabriele Burger (Leica Microsystems GmbH)). As already mentioned above, we observed the best fluorescence signal when using the 561 nm laser light, whereas an excitation with 488 nm laser light caused only weak Congo red fluorescence in the stained structures. While working with other microscope systems equipped with additional laser lines we also found very strong fluorescence of the Congo red stained structures when using 543 nm as excitation wavelength. Accordingly, the excitation maximum of Congo red stained chitin and collagen seems to be in the range between 543 nm and 561 nm. At the beginning of our study, it happened several times that the preparations were not washed thoroughly enough and remaining solute Congo red entered the glycerine. This solute Congo red did not fluoresce when exposed to 543 nm and 561 nm laser light, whereas the application of 488 nm laser light caused considerable fluorescence. This implies that the excitation maximum of 490 nm mentioned above refers to soluble Congo red, and that the fluorescence properties of Congo red change when the dye intercalates between the chitin chains and the collagen fibres.
In many small crustacean species, the exoskeleton possesses only a weak autofluorescence, which is not sufficient for an effective visualization of the external morphology (e.g. Figs 2(c) and (d)). In such cases, the fluorescence staining by Congo red strongly improves the quality of the results and thus enables an efficient visualization of the respective structures (Fig. 2(e)). The same holds true for the polychaete cuticle, which we could effectively visualize only after Congo red staining (Fig. 3(f)). However, in single cases, when the exoskeleton of the crustaceans was rather thin, even Congo red staining yielded no absolutely satisfying results. For example, after the application of the staining procedure described above the fluorescence signal of the thin exoskeleton of the decapod larva shown in Fig. 3(e) was relatively weak. In order to get reasonably good results the CLSM detector gain had to be increased considerably (see Table 1). But still the properties of the final maximum intensity projection are not very convincing as tiny structures were not visualized efficiently enough to get a clear image of them. Furthermore, as a result of the high detector gain, there is much background noise on the image. In some other cases, we observed that just certain parts of the exoskeleton such as bristles or the tips of setae are only weakly stained by Congo red. Larger parts of these structures seem to consist mainly of epicuticula, which contains no chitin and is thus not stained by Congo red (Schmidt, 1954). In order to visualize these structures, we have often found the application of Congo red fluorescence in combination with autofluorescence of the exoskeleton, the latter excited by 488 nm laser light, to be a considerable improvement. Apart from these exceptions, the application of Congo red for exoskeleton staining in small crustaceans turned out to be very successful providing excellent results, which are comparable to those described for the utilization of other efficient fluorescence dyes (Carotenuto, 1999; Zupo & Buttino, 2001; Buttino et al., 2003; Maruzzo et al., 2009) and intense autofluorescence (Michels, 2007).
The evaluation of Congo red staining revealed that, as a fluorescence marker, this dye has a very strong potential for morphological imaging of the exoskeleton of small crustaceans and the polychaete cuticle. It efficiently stains chitin and collagen in these tissues and causes an intensive fluorescence of the stained structures, which thus can be visualized by CLSM in great detail. Compared to other fluorescence dyes, Congo red is very stable. Furthermore, the excitation maximum of the structures stained with Congo red is in a range, which is covered by the laser lines of most of the confocal laser scanning microscopes available to a large number of scientists. These advantages make Congo red staining a method of choice for the visualization of the external morphology of small crustaceans and polychaetes.
We are grateful to Maria Isabel Criales, Kai Horst George, Christoph Held, Sabine Schückel, Ulrike Schückel, Gritta Veit-Köhler and Katina Spanier for providing specimens. Anneke Weber made the linguistic revision of the manuscript. Jan Michels got financial support from the virtual institute ‘PlanktonTech’ of the Helmholtz Society.