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Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456
D.H. Szarowski
J.N. Turner
New York State Department of Health, Wadsworth Center, Empire State Plaza, Albany, New York 12237
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ABSTRACT |
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Two fluorophores, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, not heretofore reported upon are described as useful dyes of fungal cell walls, septa and bud scars examined microscopically. The dyes, depending on the filter sets used, yield fluorescently stained material generally in the blue to green and yellow to red wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, respectively. They provide an excellent alternative to the more commonly used fluorophore, Calcofluor White M2R. The two fluorophores, in addition to being used at various spectral wavelengths from mercury arc sources, can be used with laser sources providing 488 nm and 543 nm line wavelengths, common to most scanning confocal microscopes. Unlike Calcofluor, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B do not fade quickly when exposed to selected light wavelengths; however, like Calcofluors they are compatible with living fungal cells.
Key words: appresoria, Calcofluor, confocal laser scanning microscopy, fluaorophore, septa
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Such brighteners often are added to commercial laundry detergents to maintain a bright appearance of washed items. The biological application of Calcofluor White M2R first was reported by Darken (1961, 1962) as a dye useful for viewing cell walls of fungi and bacteria. Since then it has been used as a fluorescent dye to view cell walls of many genera of fungi, bacteria, algae and higher plants (Albani [2001], Allen et al [1991], Apoga and Jansson [2000], Butt et al [1989], Hughes and McCully [1975], Kwon and Hoch [1991], Lloyd et al [2001], Monheit et al [1986], Pringle [1991], Santos and Snyder [2000]). The specific wall component stained has not been resolved with certainty, although it clearly binds to cell walls that are composed of chitin, cellulose and other B-1,4-linked carbohydrates (Hughes and McCully 1975, Maeda and Ishida 1967). Calcofluor White M2R, a product of the American Cyanamid Company no longer is manufactured by that company; however, the same chemical formulation is available from various vendors under different names (e.g. Fluorescent brightener 28, Fluostain I). It is important to note that cells of many fungi, bacteria and other species can grow in the dye at levels that apparently are not toxic, yet at levels that stain the cell wall with brilliant fluorescence.
Calcofluor White M2R, when used with wide-field fluorescence microscopy, is a useful dye to address biological processes in fungi and other organisms; however, it does have limitations when confocal laser scanning microscopy is considered. It is important to note that the wavelength at which it is excited is not one common to lasers (e.g. Argon 488 nm, Green HeNe 543 nm) incorporated in most confocal scanning microscope systems. Lasers that produce wavelengths of light in the UV and near-UV are available and could be used to excite the fluorophores; however, their cost remains prohibitive to applications in most biological laboratories. Furthermore such lasers generally have a relatively short lifetime, making their expense even more relevant. Calcofluor White M2R can be excited and used in multiphoton imaging systems, but the cost of these systems is high and few are present currently in biological laboratories. Spinning-disk confocal microscope systems that use mercury-arc lamps as the light source certainly can be used to image Calcofluor-stained material. A disadvantage is the short fluorescence life of Calcofluor White M2R, no matter which system is used. While the dye initially yields intense fluorescence, it fades rapidly over a relatively short time, in part due to the wavelength of light used to excite the fluorophore and in part to the composition of the wall material available for binding the dye. Similarly two other dyes occasionally used as fungal cell-wall fluorophores, Congo Red and primulin (Banuett and Herskowitz 2002, Bulawa 1993, Pringle et al 1989, Roncero and Duran 1985) fade rapidly when exposed to excitation wavelengths and for that reason generally are not used for these purposes.
A search was made for a substitute dye that could be used to view fungal cell walls with wide-field fluorescence microscopy as well as Argon or Green HeNe lasers for confocal scanning microscopy. Like Calcofluor White M2R, it was preferred that the dye not be toxic to the cells. This paper reports on two dyes, Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B, that meet those criteria; furthermore, the intensity of their fluorescence was more durable than that noted for Calcofluor White M2R. In addition they are dyes that respectively fluoresce green and red, allowing them to be used with a broader array of contrasting fluorophores than Calcofluor alone.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Culture and growth conditions.— –
A number of fungal isolates were used to evaluate wall staining by the fluorescent dyes, including: Alternaria brassicicola (Schwein.) Wiltshire, Phyllosticta ampelicida (Engelman) van der Aa, strain PY3 (ATCC No. 200578), Saccharomyces cerevisiae Meyen ex E.C. Hansen, Phytophthora infestans (Mont.) de Bary, Pythium ultimum Trow, Uromyces appendiculatus (Pers. : Pers) Unger., Colletotrichum graminicola (Cesati) Wilson, Botrytis cinerea Pers. : Fr., Magnaporthe grisea (Hebert) Barr, Rhizoctonia solani Kühn, Thielaviopsis basicola (Berk. & Broome) Ferraris, Sclerotinia sclerotiorum (Lib.) de Bary, and Gaeumannomyces graminis (Sacc.) Arx & Olivier var. tritici J. Walker. In general spores from conidiating cultures (P. ampelicida, C. graminicola, B. cinerea, M. grisea, A. solani and T. basicola) were harvested by flooding the cultures with distilled water, diluting the spore suspension to an appropriate concentration and depositing a 50 µL drop of the spore suspension on cover glasses rendered hydrophobic with either N-octadecyltrichlorosilane or dimethyldichlorosilane (Kuo and Hoch 1996) or by spin-coating a thin layer of solvent dissolved polystyrene onto the cover glasses (Kuo and Hoch 1996). Once the spores (except those of P. ampelicida) settled onto and attached to the surfaces, the water was replaced with potato-dextrose broth (PDB) (Difco, Detroit, Michigan). Spores of P. ampelicida were left to germinate in the original water drop (Kuo and Hoch 1996). Urediniospores of U. appendiculatus were deposited on the cover glasses and germinated in distilled water according to Corrêa and Hoch (1993). Ascospores of S. sclerotiorum were collected on similar cover glasses held over apothecia during induced ascospore discharge, similar to the process described by Steadman and Cook (1974). The attached ascospores were germinated in a drop of PDB. Small mycelial colonies (18 h old) of P. ultimum were grown from transferred hyphal tips in PDB or by germinating oospores in PDB. For G. graminis, small mycelial colonies were grown from hyphal fragments obtained from PDB-grown cultures processed in a blender. These mycelial fragments were allowed to develop into colonies 24–40 h in PDB on hydrophobic cover glasses, surfaces to which they attached and grew. Cells of S. cerevisiae were grown from commercially available baker’s yeast in PDB 2–4 h after which they were deposited on and adhered to poly-L-lysine treated cover glasses. Conidia of P. infestans were harvested from culture plates and similarly deposited onto poly-L-lysine treated cover glasses. All species were grown at 20 C. Several of the plant pathogenic fungal species developed appresoria.
Staining and observation.— – Microscopic observations were made with an Olympus BX-61 microscope using either the wide-field epifluorescence optics mode or the interfaced confocal laser scanning system (CLSM) (Olympus Fluoview FV-300, Melville, New York). For epifluorescence, the following Olympus filter combinations were used. For Calcofluor White M2R: excitation filter (BP360–370), dichroic mirror (DM400), and emission filter (BA420–460); For Solophenyl Flavine 7GFE 500: excitation filter (BP470–490), dichroic mirror (DM505) and emission filter (BA510–550); for Pontamine Fast Scarlet 4B: excitation filter (BP530–550), dichroic mirror (DM570) and emission filter (BA590). Two additional filter combinations obtained from Chroma Technology Corp. (Rockingham, Vermont) also were used that more accurately matched the optimal wavelengths for Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B. They were: excitation filter (360/40), dichroic mirror (400DCLP) and emission filter (500/100M); and excitation filter (HQ546/12x), dichroic mirror (Q560LP), emission filter (HQ585/40m), respectively. For CLSM, 10 mW Argon (488 nm) and 1.0 mW Green Helium Neon (Green HeNe) (543 nm) lasers were used for cell walls stained with Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B. For the 488 nm line laser, either a 510 nm long pass and/or 530 nm short pass emission filter was used, while a 605/45 band pass emission filter was used with the 543 nm laser line. Laser lines were not available to excite cells stained with Calcofluor White M2R. The Argon laser generally was used at 10% or less of full power and the Green HeNe laser at 10–30% of full power. Confocal images were captured with the Fluoview system software and subsequently transferred to Photoshop (Adobe Systems Inc., San Jose, California) for further processing.
The fluorescent dyes (0.001–0.1% of the stock solutions) were added to the medium surrounding nonfixed fungal cells for 1–5 min, after which the dye was rinsed away with either distilled water, 50 mM carbonate-bicarbonate buffer (pH 9.4) or PDB. Cover glasses bearing the stained cells were mounted, cell-side down, on the top of a stainless steel microscope specimen holder with a permanently mounted bottom cover glass (Kuo and Hoch 1996). To assess fungal growth in the dyes as well as staining of cell walls under these conditions, the dyes were added to the growth medium at the onset of growth. Later the dye was rinsed away with dye-free medium and the specimens similarly mounted as above for observation.
Photobleaching of the dyes was assessed from stained conidial walls of C. graminicola exposed to continuous UV-filtered light at wavelengths corresponding to the excitation spectra used for the specific dyes. Cytoplasm-free conidial wall preparations were obtained from conidia processed in a Mini-Bead Beater (Biospec Products, Bartlesville, Oklahoma) for 45 s (3x) and repeatedly centrifuged and resuspended in water until the preparation appeared as a homogenous conidial-wall preparation. The wall preparation appeared as mostly intact outlines of conidia bearing one or more "fractures" through which the cytoplasm had been expelled. The conidial-wall preparation was divided into equal aliquots of unstained, stained with Solophenyl Flavine, Pontamine Fast Scarlet and Calcofluor for 5 min. All dyes were diluted in 50 mM carbonate-bicarbonate buffer (pH 9.4) to 0.1% of the stock solutions. Unbound dye was removed by repeated centrifugation and rinsing with additional buffer. A series of time-lapse images of the fluorescent conidial wall preparations were captured at 5 s intervals with a SPOT-RT digital camera (Diagnostic Instruments Inc., Sterling Heights, Michigan) and processed with MetaMorph Imaging System 6.1 (Universal Imaging Corp., Downingtown, Pennsylvania). For time-course measurements, fluorescence intensity was averaged from 10 20 x 20 pixel regions (encompassing only wall areas) for each of three different preparations with the selected region of interest having an average initial gray level between 250 and 255. The data were transferred to and plotted in Excel (Microsoft, Redman, Washington) with final figures prepared with Canvas (Deneba Systems, Miami, Florida).
Assessment of pH, formaldehyde fixation and dye concentration on fluorescence were made with similar approaches as those described for photobleaching above, except temporal data were not collected. Instead single images were captured and processed with MetaMorph. Regions (280 x 480 pixels) encompassing 10–20 spores were selected in which the background gray level values 
100 were excluded, meaning only fluorescing wall material was captured and processed. In this way, "black" (e.g. 0 gray level value) or "near-black" regions outside the spore wall would not be considered as part of the averaging values. Such background gray level values would not change significantly and thus would have skewed the real fluorescent values had they been incorporated. The data were transferred to and plotted in Excel. For each treatment, the maximum gray level value was converted to 100% with the other values for the particular dye-stained wall and scaled accordingly. For each dye set of stained wall preparations, the signal detection settings of the camera remained unchanged. For effects of pH, spore walls stained with the fluorophores were rinsed and mounted in 50 mM potassium monophosphate-diphosphate buffer at either pH 5.8 or 7.0 or in 50 mM carbonate-bicarbonate buffer (pH 9.4). To assess the effects of formaldehyde fixation on the spore walls and their ability to bind the dye after fixation, the wall preparations first were fixed in 3.0% formaldehyde for 30 min (Hoch and Staples 1983), rinsed with distilled water and finally with 50 mM carbonate-bicarbonate buffer. Spore-wall preparations processed without formaldehyde served as controls. Spore wall lots were stained and rinsed with the same dye preparation (0.1% of stock solution). To determine the relative minimal concentration of dye that could be used to stain spore walls, a concentration series (0.001–0.1% of the stock solution) of each dye was prepared in 50 mM carbonate-bicarbonate buffer (pH 9.4).
Absorption and emission spectra.— – They were determined for the three fluorescent dyes with a Beckman 640 spectrometer and a Perkin-Elmer LS50B luminescence spectrometer, respectively. The dyes were diluted in 50 mM carbonate-bicarbonate buffer (pH 9.4) to concentrations appropriate for the detection range of the spectrometers.
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RESULTS |
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Absorption and emission spectra..
Solophenyl Flavine exhibited maximum absorption at 390 nm (FIG. 1). Pontamine Fast Scarlet absorbed light over a broad maximum peak (495–545 nm) with a maximum at 510 nm (FIG. 1). It is important to note that Pontamine Fast Scarlet has significant absorption at both 488 and 546 nm, wavelengths at which there are intense Hg-lines as well as wavelengths for commonly used argon and Green HeNe lasers. For comparison to Solophenyl Flavine and Pontamine Fast Scarlet, the absorption spectrum for Calcofluor also was determined; maximum absorption for this dye was 347 nm. Emission spectra were determined for the three dyes using selected excitation wavelengths, generally corresponding to the determined maximum absorption peak, nearby prominent Hg-lines or common laser line wavelengths (FIG. 1). Calcofluor excited at 365 nm (within a region of strong Hg spectral emission) exhibited a maximum emission at 440 nm. Solophenyl Flavine exhibited maximum emission at 490 nm when excited at 365 nm, whereas Pontamine Fast Scarlet exhibited maximum emission near 575 nm when excited at wavelengths of 404–546 nm. Emission spectra for two of these excitation wavelengths, 500 and 546 nm are provided (FIG. 1). The peak emission intensity when excited at 546 nm is more than twice the intensity when the dye is excited at 500 nm. However the peak wavelength for the emission spectrum for the former is close to the excitation wavelength.
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). In addition CLSM was used similarly to view cell walls in 12 fungi; however only Solophenyl Flavine and Pontamine Fast Scarlet were used because a laser with appropriate wavelength was not available for Calcofluor (FIG. 3). For wide field fluorescence observations, three fungal genera, S. cerevisiae, P. infestans and S. sclerotiorum, were imaged with filter combinations generally used for DAPI (Calcofluor), FITC (Solophenyl Flavine) and Rhodamine (Pontamine Fast Scarlet) (FIG. 2). In addition the three dyes were imaged with filter combinations that more closely match the excitation and emission spectra of the fluorophores. These images were captured with Ektachrome color film then digitized; thus the colors of the fluorescence approximates the color as viewed with the various filter combinations. All three dyes provided brightly fluorescent cell walls, septa and bud scars (the latter for S. cerevisiae). There appeared to be little difference in the wall regions or structures stained by the three dyes. Cell walls stained with Calcofluor were initially intensely bright; however the intensity diminished much faster than did either Solophenyl Flavine- or Pontamine Fast Scarlet-stained walls (see Photobleaching below). Septa were stained prominently with all three fluorophores (FIG. 2); Solophenyl Flavine- and Pontamine Fast Scarlet-stained septa are depicted for the filamentous fungi observed with CLSM (FIG. 3). In addition to walls, septa and bud scars, the extra-cellular matrix that often is prominent around germinated spores and appresoria (Kuo and Hoch 1995, O’Connell 1991, Sugui et al 1998) was demarcated generally well with the two fluorophores as shown for P. ampelicida (FIG. 3).
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). These latter dyes retained 78 and 86%, respectively of their initial fluorescence intensity over the same period. Because Solophenyl Flavine also fluoresced well when excited at 360 nm (wavelength used for Calcofluor) it was examined for photobleaching at this wavelength. It faded faster when excited at this wavelength than at 485 nm, although not to the degree that Calcofluor faded. Cellulose (Whatman No. 2 filter paper) stained with the three dyes exhibited similar fading characteristics seen in the fungal cell walls (data not shown).
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). All three fluorophores exhibited decreased fluorescence at lower pH values. Fluorescence intensity at pH 5.8 was 75–80% of the intensities observed at pH 9.4 for the three fluorophores.
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0.001% concentrations. Both Solophenyl Flavine 7GFE 500 and Pontamine Fast Scarlet 4B permitted normal growth of several tested fungal species, including U. appendiculatus, S. sclerotiorum, P. ampelicida and C. graminicola at concentrations of 0.1% (highest concentration tested) or less. Growth rates and cell morphology of these species appeared similar to cells grown in media without the dyes. Examination of the stained living fungal cells with either CLSM or wide field fluorescence microscopy exhibited intense fluorescence of the cell walls and septa (not shown). Normal spore germination and cell growth of U. appendiculatus occurred in Calcofluor at concentrations of 0.1% (0.1 mM) or less; however at 0.1% appressorium development was delayed (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fluorescence intensity of the three fluorophores was dependent on several factors, including fluorophore concentration, pH of the surrounding medium, filter combination, spectral intensity at a particular wavelength (especially applicable to Hg arc lamps) and whether the cells were left native (non-fixed) or were fixed with formaldehyde. Calcofluor was frequently a more intense (brighter) fluorophore than Solophenyl Flavine and Pontamine Fast Scarlet when the standard microscope filter sets were used. This is largely because the filter set is matched to Calcofluor’s excitation spectrum; however, when the latter two dyes were matched with more optimized filter sets, they too exhibited intense fluorescence. Phytophthora infestans and P. ultimum cells with cellulosic wall components generally fluoresced more intensely with any of the three dyes than did fungal species with chitinous walls; however there was more than sufficient brightness with the dyes under all conditions. Differences, if any, in fluorescence intensity between the fluorophores (FIG. 2
) cannot be readily assessed from these micrographs because they were obtained with automatic exposure settings. However yeast cell-wall bud-scars of S. cerevisiae nearly always stained more intensely when brightened with Solo-phenyl Flavine and Pontamine Fast Scarlet compared with Calcofluor. In addition background fluorescence was frequently less intense (darker) with the former two fluorophores.
Fluorescence intensity of Calcofluor diminished rapidly with exposure to the near-UV filtered light (FIG. 4). Such fading of the fluorophore often has been reported, but because the initial intensity was sufficiently high (for image capture purposes) this generally has not been a serious problem (Hughes and McCully 1975, Pringle 1991). In part the rapid fading of Calcofluor compared to the other two fluorophores reported herein likely is due to a number of factors: shorter wavelength (and thus greater energy) of light transmitted by the excitation filters (ca. 360 nm vs. >400 nm), as well as the dichroic filter, different overall intensities of the light at the different wavelengths (e.g. there is a very strong mercury line at 365 nm but not at 488 nm) and certainly a difference in chemical structures. The pH of the medium clearly influenced the fluorescence intensity of the wall-bound fluorophores (FIG. 5). This was not unexpected because it often has been reported that the Calcofluor was brighter at higher pH values (Darken 1962, Gull et al 1972, Hughes and McCully 1975).
It was somewhat surprising that Solophenyl Flavine yielded excellent fluorescence when excited with filtered Hg-light of 470–490 nm, as well as the 488 nm laser line, because both of these spectral wavelengths are at the tail end of the absorption spectrum for this dye. This is especially surprising because there is relatively little spectral intensity in this range; the laser intensity could have been increased, although it generally was kept attenuated at or below 10% full power. Both light sources, however, captured most of the emission wavelength (ca. 510 nm and above). Excitation (340–380 nm) of Solophenyl Flavine with the customized filter set clearly yielded more optimal fluorescence in the wide field microscope. For example, using uniform conditions (same Solophenyl Flavine-stained spore-wall preparation, image size, etc.) it took ca. 40 ms to capture a representative image using the standard-FITC filter set and only ca. 5 ms with the customized filter set (data not shown). Because the original standard-rhodamine filter set was already closely matched to Pontamine Fast Scarlet there was less difference in the image capture time with the customized filters (18–25 ms vs. 12–15 ms, respectively).
Calcofluor is used as a vital dye in many cell systems; however at higher concentrations it sometimes becomes toxic influencing assembly of cellulose and chitin fibrils (Bulawa 1993, Haigler et al 1980, Roncero and Duran 1985). We too noted in this study that concentrations higher than 0.1% slowed germ tube growth and delayed appressorium maturity. Solophenyl Flavine and Pontamine Fast Scarlet at similar concentrations did not appear to be inhibitory, although the actual concentration of active ingredients in Pontamine Fast Scarlet is not known because it is supplied as a premixed liquid.
Viewing fungal cell walls, septa, bud scars, appresorial lobes, etc., as well as similar components of other cell-types, is important to morphological and cell biological studies. Solophenyl Flavine and Pontamine Fast Scarlet, along with the commonly used fluorophore Calcofluor, expand the selection of fluorophores available for use in these studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: hch1{at}cornell.edu
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) matches the standard microscope filter set, and the lighter band (
) matches the customized filter set. Also noted are the laser lines of 488 and 543 nm.
