Histochemistry 64, 115-118 (1979)
Histochemistry 9 by Springer-Verlag 1979
Fluorescent Colloidal Gold: A Cytochemical Marker for Fluorescent and Electron Microscopy M. Horisberger and Monique Vonlanthen Nestl6 Research Department, P.O. Box 88 CH-1814 La Tour-de-Peilz,Switzerland
Summary. The gold method was further developed for fluorescent microscopy. Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin. The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA. By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking. Introduction In recent years, various cytochemical markers have been developed for either photonic or electron microscopy. However, few of them are applicable simultaneously at both low and high resolution levels. Latex spheres tagged with fluorescent molecules have been used as markers for fluorescent microscopy and scanning electron microscopy (Molday et al., 1975). More recently lectins were detected on cell surfaces by fluorescent derivatives of glycosylated ferritin (Kieda et al., 1979). Colloidal gold can be labelled with a variety of macromolecules and has been used as an electron dense marker in both the transmission (Faulk and Taylor, 1971 ; Horisberger and Rosset, 1977; Geoghegan and Ackerman, 1977; Roth and Binder, 1978) and the scanning electron microscope (Horisberger and Rosset, 1977). In this study, we have further developed the gold method for fluorescent microscopy. Mannan was detected on the yeast cell surface by fluorescent derivatives of Concanavalin A and avidin either by the one-step, or by the two-step method via a biotinyl derivative of ConA (Bayer et al., 1976). The marked cells were examined by fluorescence microscopy and transmission electron microscopy. Materials and Methods Materials. ConcanavalinA (ConA) was from Miles Laboratories, biotinyl-ConA and rhodamine avidin from Vector Laboratories, Carbowax 20-M (polyethylene glycol compound) from Union
0301-5564/79/0064/0115/$01.00
116
M. Horisberger and M. Vonlanthen
Carbide Chemicals Co. and chloroauric acid HAuC14 aq. (50% Au, purum) from Fluka AG (Buchs, Switzerland). Saccharomyces cerevisiae (baker's yeast, laboratory strain) was grown as described earlier (Horisberger and Vonlanthen, 1977). ConA was conjugated to tetramethylrhodamine isothiocyanate (TRITC) as described by Roth et al. (1978). Buffer A was 0.15 M NaC1-0.02 M Tris, pH 7.4; buffer B was buffer A containing in addition 0.5 mg/ml Carbowax 20-M and sodium azide and made 0.001 M in CaClz and MnC12. All Carbowax 20-M containing solutions were filtered through Millipore (0.45 gm pore size) (Horisberger and Rosset, 1977). Labelling of Gold Granules. Gold granules having 12nm in size ( A u l 2 , 100ml) were prepared by the reduction of gold chloride with sodium ascorbate (Stathis and Fabrikanos, 1958) as modified by Horisberger and Vonlanthen (1978). Au12 granules (50 ml) were labelled with TRITC-ConA (I.18 mg) and TRITC-avidin (0.225 rag) by the procedure described elsewhere (Horisberger and Vonlanthen, 1978). The colloids were recovered by centrifugation and resuspended in buffer B. Marking of Saccharomyces cerevisiae Cells'. The yeast cells were washed in buffer A (3 • ) and suspended in buffer B (A65 onm 1.0). (a) one-step method: the cells (0.5 ml) were incubated for 2 h at 20~ C with -fRITC-ConA-AuI2 (A520 nm 1.2, 0.5 ml). The cells were washed twice with buffer B. A control was performed in the presence of 50 mg methyl-c~-l>mannopyranoside. (b) two-step method: the cells (0.5 ml) were incubated with biotinyl-ConA (0.5 mg in 0.5 ml buffer B) for 2 h at 20~ C. A control was performed in the presence of 50 mg methyl-c~-o-mannopyronoside. The cells were washed with buffer B (3 x ) and incubated with TRITC-avidin-Au12 (A520 nm 5.0) for 30 rain at 20~ C. The cells were washed twice with buffer B. The marked cells and the controls were then examined by fluorescent microscopy (Orthoplan Leitz) and transmission electron microscopy (Philips EM 300). Longer incubation time did not affect the results. For electron microscopy, the cells were fixed for 15 h at 4~ C in 0.1 M phosphate buffer, pH 7 containing 2% OsO~. They were then washed with the buffer and dehydrated in 30% alcohol. Finally the cells were stained for 15 h in 50% ethanol containing 2_5% uranyl acetate and embedded according to Spurr (1969).
Results and Discussion M a r k i n g of S. cerevisiae cells with T R I T C - C o n A - A u 1 2 (one-step m e t h o d ) led to a red-orange fluorescence (Fig. l a a n d b) i n d i c a t i n g the presence of cell surface m a n n a n (Horisberger a n d Rosset, 1977; Horisberger a n d V o n l a n t h e n , 1977). I n the controls, fluorescence was completely absent. These results d e m o n strate that gold granules can be labelled with r h o d a m i n e conjugates a n d used successfully in fluorescent microscopy. However, a m o r e brilliant fluorescence was exhibited by the two-step m e t h o d (Fig. 1 a) t h a n by the one-step m e t h o d (Fig. 1 b). The o b s e r v a t i o n s were confirmed by e x a m i n i n g t h i n sections of m a r k e d cells by t r a n s m i s s i o n electron microscopy. I n the one-step m e t h o d (Fig. l c ) the m a r k e r was sparsely distributed a n d f o u n d in aggregates. As shown earlier (Horisberger a n d Rosset, 1977), this is due to a c o n t i n u o u s leakage of traces of m a n n a n , causing a n aggregation of the marker. By the two-step m e t h o d (Fig. 1 d), m a r k i n g was denser a n d m o r e u n i f o r m . D u e p r o b a b l y to a fixation of cell wall m a n n a n by b o u n d b i o t i n y l - C o n A , the m a r k e r was m u c h less aggregated t h a n by the one-step method. The distances observed between the yeast cell wail a n d the m a r k e r is a t t r i b u t e d to the size of b i o t i n y l - C o n A molecules since this spacing was not observed by the other m e t h o d . t n c o n c l u s i o n these results d e m o n s t r a t e : (a) that gold granules tagged with
Fluorescent Colloidal Gold
117
Fig. 1. a Saccharomyces cerevisiae cell wall m a n n a n marked with TRITC-avidin-Au12 granules by the two-step method. Fluorescence is strong, b Marking the cells by the one-step method with TRITC-ConA-Au12 results in a weak fluoresceilce, e Cells marked as under b and examined by transmission electron microscopy. Note the presence of aggregates. Marking is sparse, el Cells marked as under a. Marking is more uniform and dense. Some aggregates are still present. Note the u n m a r k e d spacing between the cell wall and the marker. Fig. a and b: x 680; e and d: x 15,000
118
M. Horisberger and M. Vonlanthen
fluorescent probes can be successfully used both in fluorescent and electron microscopy; (b) the method is specific; (c) the two-step method leads usually to a more dense marking than the one-step method especially when cellular components leak form a cell (Horisberger, 1979); (d) affinity cytochemistry via the avidin-biotin complex (Heitzmann and Richards, 1974; Bayer et al., 1976) is feasible by the gold method; (e) the gold method alleviates the time consuming chemical coupling of probes to ferritin; (f) the advantage of the fluorescent gold method resides in the possibility to optimize the conditions of marking by observing the fluorescence, before examination by electron microscopy. Acknowledgment. The authors thank Mrs. M. Weber for the photographic work. References Bayer, E.A., Skutelsky, E., Wynne, D., Wilchek, M.: Preparation of ferritin avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry. J. Histochem. Cytochem. 24, 933-939 (1976) Faulk, W.P., Taylor, G.M. : An immunocolloid method for the electron microscope. Immunochemistry 8, 1081-1083 (1971) Geoghegan, W.D., Ackerman, G.A. : Adsorption of horseradish peroxidase, ovomucoid and antiimmuno-globulin to colloidal gold for the indirect direction of Concanavalin A, wheat germ agglntinin and goat anti-human immunoglobulin on cell surfaces at the electron microscopic level: a new method, theory and application. J. Histochem. Cytochem. 25, 1187-1200 (1977) Heizmann, H., Richards, FM. : Use of the avidin-biotin comptex for specific staining of biological membranes in electron microscopy. Proc. Natl. Acad. Sci. USA 71, 3537-354I (1974) Horisberger, M. : Evaluation of coiloidal gold as a cytochemical marker for transmission and scanning electron microscopy. Biol. Cell. 36 (1979, in press) Horisberger, M., Rosset, J. : Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295-305 (1977) Horisberger, M., Vonlanthen, M.: Location of mannan and chitin on thin sections of budding yeasts with gold markers. Arch. Microbiol. 115, 1-7 (1977) Horisberger, M., Vonlanthen, M. : Simultaneous localization of an hepatic binding protein specific for galactose and of galactose-containing receptors on rat hepatocytes, J. Histochem. Cytochem. 26, 960-966 (1978) Kieda, C., Roche, A.C., Delmotte, F., Monsigny, M. : Lymphocyte membrane lectins. Direct visualization by the use of fluoresceinyl-glycosylated cytochemical markers. FEBS Lett. 99, 329-332 (1979) Motday, R.S., Dreyer, W.J., Rembaum, A., Yen, S.P.S. : New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy. J. Cell Biol. 64, 75-88 (1975) Roth, J., Binder, M.: Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques. J. Histochem. Cytochem. 26, 163-169 (1978) Roth, J., Binder, M., Gerhard, U.J.: Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components. Histochemistry 56, 265-273 (1978) Spurr, A.R. : A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Stathis, E.C., Fabrikanos, A.: Preparation of colloidal gold. Chem. Ind. (London) 27, 860 861 (1958)
Received July 16, 1979
Histochemistry 9 by Springer-Verlag 1979
Fluorescent Colloidal Gold: A Cytochemical Marker for Fluorescent and Electron Microscopy M. Horisberger and Monique Vonlanthen Nestl6 Research Department, P.O. Box 88 CH-1814 La Tour-de-Peilz,Switzerland
Summary. The gold method was further developed for fluorescent microscopy. Gold granules (12 nm in size) were labelled with rhodamine conjugates of Concanavalin A and avidin. The fluorescent markers were used to mark cell wall mannan on the yeast Saccharomyces cerevisiae either by the one-step, or by the two-step method via a biotinyl derivative of ConA. By fluorescence or transmission electron microscopy, the two-step method was found to achieve a higher density of marking. Introduction In recent years, various cytochemical markers have been developed for either photonic or electron microscopy. However, few of them are applicable simultaneously at both low and high resolution levels. Latex spheres tagged with fluorescent molecules have been used as markers for fluorescent microscopy and scanning electron microscopy (Molday et al., 1975). More recently lectins were detected on cell surfaces by fluorescent derivatives of glycosylated ferritin (Kieda et al., 1979). Colloidal gold can be labelled with a variety of macromolecules and has been used as an electron dense marker in both the transmission (Faulk and Taylor, 1971 ; Horisberger and Rosset, 1977; Geoghegan and Ackerman, 1977; Roth and Binder, 1978) and the scanning electron microscope (Horisberger and Rosset, 1977). In this study, we have further developed the gold method for fluorescent microscopy. Mannan was detected on the yeast cell surface by fluorescent derivatives of Concanavalin A and avidin either by the one-step, or by the two-step method via a biotinyl derivative of ConA (Bayer et al., 1976). The marked cells were examined by fluorescence microscopy and transmission electron microscopy. Materials and Methods Materials. ConcanavalinA (ConA) was from Miles Laboratories, biotinyl-ConA and rhodamine avidin from Vector Laboratories, Carbowax 20-M (polyethylene glycol compound) from Union
0301-5564/79/0064/0115/$01.00
116
M. Horisberger and M. Vonlanthen
Carbide Chemicals Co. and chloroauric acid HAuC14 aq. (50% Au, purum) from Fluka AG (Buchs, Switzerland). Saccharomyces cerevisiae (baker's yeast, laboratory strain) was grown as described earlier (Horisberger and Vonlanthen, 1977). ConA was conjugated to tetramethylrhodamine isothiocyanate (TRITC) as described by Roth et al. (1978). Buffer A was 0.15 M NaC1-0.02 M Tris, pH 7.4; buffer B was buffer A containing in addition 0.5 mg/ml Carbowax 20-M and sodium azide and made 0.001 M in CaClz and MnC12. All Carbowax 20-M containing solutions were filtered through Millipore (0.45 gm pore size) (Horisberger and Rosset, 1977). Labelling of Gold Granules. Gold granules having 12nm in size ( A u l 2 , 100ml) were prepared by the reduction of gold chloride with sodium ascorbate (Stathis and Fabrikanos, 1958) as modified by Horisberger and Vonlanthen (1978). Au12 granules (50 ml) were labelled with TRITC-ConA (I.18 mg) and TRITC-avidin (0.225 rag) by the procedure described elsewhere (Horisberger and Vonlanthen, 1978). The colloids were recovered by centrifugation and resuspended in buffer B. Marking of Saccharomyces cerevisiae Cells'. The yeast cells were washed in buffer A (3 • ) and suspended in buffer B (A65 onm 1.0). (a) one-step method: the cells (0.5 ml) were incubated for 2 h at 20~ C with -fRITC-ConA-AuI2 (A520 nm 1.2, 0.5 ml). The cells were washed twice with buffer B. A control was performed in the presence of 50 mg methyl-c~-l>mannopyranoside. (b) two-step method: the cells (0.5 ml) were incubated with biotinyl-ConA (0.5 mg in 0.5 ml buffer B) for 2 h at 20~ C. A control was performed in the presence of 50 mg methyl-c~-o-mannopyronoside. The cells were washed with buffer B (3 x ) and incubated with TRITC-avidin-Au12 (A520 nm 5.0) for 30 rain at 20~ C. The cells were washed twice with buffer B. The marked cells and the controls were then examined by fluorescent microscopy (Orthoplan Leitz) and transmission electron microscopy (Philips EM 300). Longer incubation time did not affect the results. For electron microscopy, the cells were fixed for 15 h at 4~ C in 0.1 M phosphate buffer, pH 7 containing 2% OsO~. They were then washed with the buffer and dehydrated in 30% alcohol. Finally the cells were stained for 15 h in 50% ethanol containing 2_5% uranyl acetate and embedded according to Spurr (1969).
Results and Discussion M a r k i n g of S. cerevisiae cells with T R I T C - C o n A - A u 1 2 (one-step m e t h o d ) led to a red-orange fluorescence (Fig. l a a n d b) i n d i c a t i n g the presence of cell surface m a n n a n (Horisberger a n d Rosset, 1977; Horisberger a n d V o n l a n t h e n , 1977). I n the controls, fluorescence was completely absent. These results d e m o n strate that gold granules can be labelled with r h o d a m i n e conjugates a n d used successfully in fluorescent microscopy. However, a m o r e brilliant fluorescence was exhibited by the two-step m e t h o d (Fig. 1 a) t h a n by the one-step m e t h o d (Fig. 1 b). The o b s e r v a t i o n s were confirmed by e x a m i n i n g t h i n sections of m a r k e d cells by t r a n s m i s s i o n electron microscopy. I n the one-step m e t h o d (Fig. l c ) the m a r k e r was sparsely distributed a n d f o u n d in aggregates. As shown earlier (Horisberger a n d Rosset, 1977), this is due to a c o n t i n u o u s leakage of traces of m a n n a n , causing a n aggregation of the marker. By the two-step m e t h o d (Fig. 1 d), m a r k i n g was denser a n d m o r e u n i f o r m . D u e p r o b a b l y to a fixation of cell wall m a n n a n by b o u n d b i o t i n y l - C o n A , the m a r k e r was m u c h less aggregated t h a n by the one-step method. The distances observed between the yeast cell wail a n d the m a r k e r is a t t r i b u t e d to the size of b i o t i n y l - C o n A molecules since this spacing was not observed by the other m e t h o d . t n c o n c l u s i o n these results d e m o n s t r a t e : (a) that gold granules tagged with
Fluorescent Colloidal Gold
117
Fig. 1. a Saccharomyces cerevisiae cell wall m a n n a n marked with TRITC-avidin-Au12 granules by the two-step method. Fluorescence is strong, b Marking the cells by the one-step method with TRITC-ConA-Au12 results in a weak fluoresceilce, e Cells marked as under b and examined by transmission electron microscopy. Note the presence of aggregates. Marking is sparse, el Cells marked as under a. Marking is more uniform and dense. Some aggregates are still present. Note the u n m a r k e d spacing between the cell wall and the marker. Fig. a and b: x 680; e and d: x 15,000
118
M. Horisberger and M. Vonlanthen
fluorescent probes can be successfully used both in fluorescent and electron microscopy; (b) the method is specific; (c) the two-step method leads usually to a more dense marking than the one-step method especially when cellular components leak form a cell (Horisberger, 1979); (d) affinity cytochemistry via the avidin-biotin complex (Heitzmann and Richards, 1974; Bayer et al., 1976) is feasible by the gold method; (e) the gold method alleviates the time consuming chemical coupling of probes to ferritin; (f) the advantage of the fluorescent gold method resides in the possibility to optimize the conditions of marking by observing the fluorescence, before examination by electron microscopy. Acknowledgment. The authors thank Mrs. M. Weber for the photographic work. References Bayer, E.A., Skutelsky, E., Wynne, D., Wilchek, M.: Preparation of ferritin avidin conjugates by reductive alkylation for use in electron microscopic cytochemistry. J. Histochem. Cytochem. 24, 933-939 (1976) Faulk, W.P., Taylor, G.M. : An immunocolloid method for the electron microscope. Immunochemistry 8, 1081-1083 (1971) Geoghegan, W.D., Ackerman, G.A. : Adsorption of horseradish peroxidase, ovomucoid and antiimmuno-globulin to colloidal gold for the indirect direction of Concanavalin A, wheat germ agglntinin and goat anti-human immunoglobulin on cell surfaces at the electron microscopic level: a new method, theory and application. J. Histochem. Cytochem. 25, 1187-1200 (1977) Heizmann, H., Richards, FM. : Use of the avidin-biotin comptex for specific staining of biological membranes in electron microscopy. Proc. Natl. Acad. Sci. USA 71, 3537-354I (1974) Horisberger, M. : Evaluation of coiloidal gold as a cytochemical marker for transmission and scanning electron microscopy. Biol. Cell. 36 (1979, in press) Horisberger, M., Rosset, J. : Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295-305 (1977) Horisberger, M., Vonlanthen, M.: Location of mannan and chitin on thin sections of budding yeasts with gold markers. Arch. Microbiol. 115, 1-7 (1977) Horisberger, M., Vonlanthen, M. : Simultaneous localization of an hepatic binding protein specific for galactose and of galactose-containing receptors on rat hepatocytes, J. Histochem. Cytochem. 26, 960-966 (1978) Kieda, C., Roche, A.C., Delmotte, F., Monsigny, M. : Lymphocyte membrane lectins. Direct visualization by the use of fluoresceinyl-glycosylated cytochemical markers. FEBS Lett. 99, 329-332 (1979) Motday, R.S., Dreyer, W.J., Rembaum, A., Yen, S.P.S. : New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy. J. Cell Biol. 64, 75-88 (1975) Roth, J., Binder, M.: Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques. J. Histochem. Cytochem. 26, 163-169 (1978) Roth, J., Binder, M., Gerhard, U.J.: Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components. Histochemistry 56, 265-273 (1978) Spurr, A.R. : A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43 (1969) Stathis, E.C., Fabrikanos, A.: Preparation of colloidal gold. Chem. Ind. (London) 27, 860 861 (1958)
Received July 16, 1979
