Planta (Berl.). 95, 341--350 (1970) 9 by Springer-Verlag 1970
Electron Probe Analysis of Freeze-Substituted, Epoxy Resin Embedded Tissue for Ion Transport Studies in Plants ANDR]~ L h v c n ~ , A~THU~ R. SPUR~ and RAr~OND W. WITTKOPP Departments of Soils and Plant Nutrition, of Vegetable Crops, and of Geology, University of California, Davis Received September 21, 1970
Summary. A new technique is described to prepare plant material for electron probe analysis. Root segments 1 mm in length were frozen at --170 ~ freeze-substituted with anhydrous ether at --30 ~ and infiltrated with Spurr's low-viscosity epoxy resin embedding medium at low temperatures. Sections 1 and 2 V thick were cut anhydrously using hexylene glycol in the ultramicrotome trough, mounted on the polished surface of a Be disc and vacuum coated with 150-200/~ aluminum. The new technique allows retention of water-soluble ions at the original sites in the tissue and is superior to eryostat sectioning in spatial resolution of electron probe analysis and in the preservation of cellular structures. The lateral transport of K + into the xylem of corn roots has been successfully studied by electron probe analysis of freeze-substituted, epoxy resin embedded material. Introduction Correlation of physiological experiments on ion transport in plants wiht structural investigations has recently gained much attention. Electron probe analysis and microautoradiography are two methods which yield direct results for localizing soluble ions transported in the plant, and mineral nutrients bound to the tissue. That these two methods supplement each other has been demonstrated in an experimental study (Li~uehli and Liittge, 1968). Mineral nutrients in the plant occur in part as solutes. During the preparation of specimens for electron probe analysis or microautoradiography, it is essential to minimize the displacement of inorganic solutes in the tissue. The techniques applicable to microautoradiography of soluble ions have been summarized by Lfittge and Weigl (1965). The eryostat technique is believed to allow ions to be retained at the original sites. Hence, it has been employed in the past to prepare plant material, for electron probe analysis (L~uehli, 1967a; Li~uehli and Ltittge, 1968; Rasmussen et al., 1968). However, cryostat sectioning of plant material does not yield thin sections and the preservation of cellular details is
342
A. L~uchli, A. R. Spurr, and R. W. Wittkopp:
poor. These are limiting factors i n achieving good spatial resolution in electron probe analysis. I n a n account of the factors t h a t determine resolution, A n d e r s e n (1967) states t h a t resolution depends on the diameter of the electron b e a m a n d on the v o l u m e of the specimen c o n t r i b u t i n g to the analysis. The volume of the sample excited is a f u n c t i o n of the depth of electron p e n e t r a t i o n a n d of electron a n d x - r a y scattering. Consequently, t h i n sections on the order of 1 ~ thickness are preferable to the 16-20 ~z sections o b t a i n e d b y the cryostat technique. I n this paper we report a new t e c h n i q u e to prepare sections of freezes u b s t i t u t e d material e m b e d d e d i n epoxy resin for s t u d y i n g ion t r a n s p o r t in p l a n t s t h r o u g h electron probe analysis.
Materials and Methods Plant Material. Seedlings of corn, Zea mays L., DeKalb 805, were grown as described by Lguchli and Epstein (1970). The seedlings were germinated in the dark for 4 days and then grown for 4 days in a growth chamber on 1/10 concentration Johnson nutrient solution (Johnson et al., 1957) but with Fe-EDTA supplied at 4/10 concentration. For the experiments, the root of an intact seedling was exposed for 3 hr at 30 ~ to an aerated solution of 0.2 mM KCI+0.5 mM CaSO4 and then excised just below the seed. A length of polyethylene tubing was attached to the root, and the root then immersed in an identical solution and allowed to exude for 1 hr. Thereafter, the root was rinsed with deionized water for 1 min and processed for electron probe analysis. Under these conditions, the translocation of K + and C1into the xylem exudate occurs at a steady rate (cf. L~,uehli and Epstein, 1970). 9 $'reezing and Freeze-Substitution. In a coldroom at 4 ~ root segments 1 mm in length were cut 10-11 mm proximal to the root apex and immediately frozen in a mixture of 8% (v/v) methyleyclohexane in isopentane cooled to --170 ~ with liquid nitrogen. The frozen tissue was dehydrated by freeze-substitution with anhydrous ether. Liittge and Weigl (1965) used ether in freeze-substitution to prepare specimens for microautoradiography and obtained results comparable to those using tissue dehydrated by freeze-drying or sectioned with a cryostat (Liittge and Weigl, 1965; Krichbaum et al., 1967). Prior to use, the ether was dehydrated with aluminum oxide (A1203, basic; Brockman, activity grade I) according to Wohlleben (1955). Four replicate root segments were transferred from the freezing mixture to dry ice and then plunged into a 25 ml polyethylene serew-cap vial containing 15 ml anhydrous ether + 2 g A1203, basic, at --30 ~ The vials were placed in a shaking device, which was operated in a low-temperature cabinet at --30 ~ and were continuously shaken for 7 days, at which time freeze-substitution was completed. Infiltration with Epoxy t~esin, and Embedding. Spurr's low-viscosity epoxy resin embedding medium E (Spurr, 1969) was chosen for infiltration and embedding. Based on the physical characteristics of medium E and its components shown in Table 1, the epoxy resin portion (vinylcyelohexene dioxide) was employed for initial infiltration into the tissue at --30 ~ the temperature used in freeze-substitution. Final infiltration was accomplished with the complete medium E starting at --12 ~ and finishing at + 4 ~. 1 Temperatures throughout the paper are in degrees centigrade (~
Electron Probe Analysis for Ion Transport Studies
343
Table 1. Physical characteristics o] Spurr's low-viscosity epoxy resin embedding
medium E and its components at low temperatures solid; -k fluid, high viscosity; -k -k fluid, medium viscosity; q- ~--k fluid, low viscosity. -
-
Temperature
Epoxy resina
Flexibilizer b
Anhydride Accelehardener c rater a
+++ §247247 §247247 §247247
+ §247 §247 §247
--§ §247
l~[edium E
(~ --30 --20 --12 + 4
+++ §247247 §247 §247
§ §247 §247247
a Vinyl cyclohexene dioxide (ERL-4206). b Diglycidyl ether of polypropylene glycol (D.E.R 736). e Nonenyl succinie anhydride (NSA). d Dimethylamincthanol (S-l). e 7.8 cP at 20 ~ ~ 60 cP at 25 ~
The freeze-substituted tissue segments were washed for 3 hr at --30 ~ with acetone dehydrated with Al203 (acid; Brockman, activity grade I) according to Wohlleben (1955). The acetone was changed every hour. Infiltration was started by adding to the last change of acetone an equal quantity of vinyl cyclohexene dioxide at --30 ~ After 4 hr the mixture was poured and drained from the vials and replaced by 100% vinylcyclohexene dioxide. Infiltration continued at --30 ~ overnight and then another change was made of the epoxy resin component before warming the vials up slowly to --12 ~ An equal quantity of the complete medium E was then added and allowed to stand until the evening. Another equal quantity of medium E was added and the vials were brought to -b4 ~ overnight. The tissue segments were then transferred to small vials containing 100% medium E and warmed to room temperature. After one additional change of the medium the specimens were transferred to oven-dried gelatin capsules containing medium E and cured for 16 hr at 70 ~ Specimen Preparation ]or Electron Probe Analysis. Polished discs of aluminum and beryllium, 2.54 cm in diameter, were used as section holders. The A1 discs were prepared essentially as described by L~uchli (1967a). Discs of pure Be with one side polished were obtained commercially (American Beryllium Corp., Inc., Sarasota, Florida). Transverse root sections 1 and 2 ~z thick were cut with a diamond knife on a Cambridge ultramicrotome. To avoid exposure of the sections to water (c/. Pease, 1966) anhydrous sectioning was accomplished through the use of hexylene glycol in the trough. The sections were removed from the surface of the glycol with a fine brush and placed on discs carrying a thin film of hexylene glycol. The discs were then placed in an oven at 70 ~ for 2 hr to spread the sections and to evaporate the hexylene glycol. When A1 discs were used ethylene glycol was employed as the fluid in the trough. J u s t prior to their use the surface of the A1 discs was pretreated with concentrated H~SO~ for about 10 rain, rinsed with deionized water, and dried in the oven to provide a surface which could be wetted with ethylene glycol. Otherwise
344
A. L/~uchli, A. R. Spurr, and R. W. Wittkopp:
the procedure was as described for Be discs. The discs with sections adhering were vacuum coated with 150-200 ~ A1. In this application, coating with A1 was found to be superior to carbon coating in that less damage was induced by the electron beam, probably because of better heat conduction. Elemental Analyses. Elements were measured with an Applied Research Laboratories electron probe x-ray analyzer, model EMX-SM, with scanning capabilities. The instrument was operated at 15 KeV accelerating voltage, 0.5 ~ electron beam diameter, and a sample current of 0.023 ~A on Be which was equivalent to 0.027 ~A on A1. Transmission Electron Microscopy. Electron micrographs from thin sections of freeze-substituted, epoxy resin embedded tissue were obtained by a slightly altered procedure. Root segments were freeze-substituted with ether as described, washed with acetone at --30 ~ and then fixed with 1% Os04 in acetone at --70 ~ overnight. At this temperature OsOa in acetone is stable (Feder and Sidman, 1958). The fixed material was washed with acetone, warmed to --30 ~ and infiltrated and embedded as described. Standard thin-sectioning procedures and contrasting with uranyl acetate and lead citrate were used. Electron micrographs obtained by this procedure will be published elsewhere (Li~uchli, Spurt and Epstein, in preparation).
Results and Discussion Measurement of b a c k g r o u n d r a d i a t i o n of Be a n d A1 are presented in Table 2. F o r analysis of K(K~) a n d Ca(K~), b a c k g r o u n d r a d i a t i o n of Be Table 2. Background radiation o/Be and A1 discs (counts per 100 sec) a
Be A1
K (Ks) (ADP)
Mg (Ks) (RAP)
Ca (Ks) (LiF)
1547 ~: 56.5 5283 • 102
944 • 26 37404 • 140.3
108 -4- 15 332 4-16
a Discs were vacuum coated with 150-200 A A1; electron probe x-ray analyzer was operated at 15 KeV with sample current of 0.023 ~A on Be which is equivalent to 0.027 ~A on Al; analyzing crystals are given below the elements analyzed (the recently introduced crystal RAP is rubidium dihydrogen phosphate); the data represent the means of 4 replicates with standard errors.
is only a b o u t 30% of t h a t of A1. The A1 we used contains Mg as a n i m p u r i t y a n d is therefore i n a d e q u a t e for the m e a s u r e m e n t of Mg i n tissue sections. Beryllium is the superior s u p p o r t i n g m a t e r i a l because a low b a c k g r o u n d r a d i a t i o n is needed to increase the peak to b a c k g r o u n d ratio in electron probe analysis. Table 3 shows d a t a on b a c k g r o u n d r a d i a t i o n of epoxy resin on Be w i t h o u t p l a n t tissue present. B a c k g r o u n d readings of K ( K ~ ) r a d i a t i o n from sections of polymerized epoxy resin on Be a n d from Be alone are n o t significantly different. However, the s t a n d a r d error of the m e a n of b a c k g r o u n d m e a s u r e m e n t s rises with a n increasing thickness of resin. The
Electron Probe Analysis for Ion Transport Studies
345
Table 3. Background radiation o/Be disc and o/epoxy resin sections (without tissue) on Be discs (counts per 100 sec)a
Be Epoxy resin (1 ~) Epoxy resin (2 ~)
K (Ks) (ADP)
Mg (Ks) (RAP)
Ca (Ks) (LiF)
C1 (Kc0 (ADP)
1551 ~ 66.9 1808:t:113 1725___254
986 :t: 55 979~ 14 986~ 165
108 • 10 101:J-10 118•
312 • 7.8 3082-[-439 5696•
a Samples were vacuum coated with 150-200A A1; electron probe x-ray analyzer was operated at 15 KeV with 0.023 ~*Asample current; analyzing crystals are given below the elements analyzed; the data represent the means of 4 replicates with standard errors.
resin is not contaminated with Mg and Ca, but it contains appreciable amounts of C1 which causes a high standard error for the background detection of CI(K~) radiation in 2 bt thick sections. Similar values of background radiation were obtained when the readings were taken in the resin-impregnated tissue and in the resinous embedding material outside of the tissue. Epoxy resins based on epichlorohydrin may eontMn small amounts of C1. Ingram and I-Iogben (1968) report that Epon 826 contains 30 millimoles kg -1 of organically bound C1. Some C1 also occurs in the ionic form in resins of this type. Vinyl cyelohexene dioxide, the principal epoxy resin employed in our study, is not derived from Cl-eontMning compounds and is presumed to have very low levels of C1. The epoxy resin flexibilizer D.E.t~. 736 in our embedding mixture is derived in part from epiehlorohydrin and averages 0.5% C1. ttowever, D.E.IK 736 is only a small part of the mixture and on the basis of the total resinous formulation the C1 content is about 0.073%. Still, this level is too high for studies in which C1 is of interest. Yet, it is possible to prepare epoxy resins with very low levels of C1. Another alternative is to consider embedding in methaerylate (Gielink et al., 1966), but GMIe and Stuve (1968) do not recommend it because of its relative instability under the electron beam. Methaerylate also has other disadvantages sueh as marked shrinkage and bubble formation during polymerization. Our preparation technique was applied to a study on the lateral transport of K + into the xylem of corn roots (Liiuehli. Spurr and Epstein, in preparation). Preliminary experiments showed that the electron probe x-ray analyzer was utilized at the limits of its detection sensitivity when K was measured in sections 1 bt thick. More aceurate data were obtained with 2 b~ sections in evaluating the pattern of K distribution in transverse sections of roots. Further, the electron beam caused less noticeable damage in 2 bt sections as compared with thicker sections. 23 ]?lant~(Berl.), ]~d.95
346
A. Lguchli, A. P~. Spurr, and R. W. Wittkopp:
Fig. I. Transverse section, 2 ~ thick, of a corn root taken 10-11 mm proximal to the root apex. Root segment frozen at --170 ~ freeze-substituted with anhych'ous ether and embedded in low-viscosity epoxy resin medium. Section mounted on a Be disc, cotaed with 150-200 A A1 and photographed by reflected-light microscopy. Section shows the effects of point analysis (PA) with central dark spot (a), secondarily excited area (b) and dark margin (c), and also the effects of a line scan (LS). Endodermis (E), pericyele (P), xylem vessel (Xy). • 720
Fig. l shows part of a transverse section, 2 ~ thick, taken 10-11 m m proximal to the root apex. The effects of exposing the section to a static electron beam for point analysis (PA) are visible at several sites. The central dark spot (a) is caused directly b y the beam and indicates the p r i m a r y spatial resolution which is better t h a n 1 ~z. There is also a secondarily excited area (b) surrounded b y a dark margin (c). The area (b) is about 7 ~z in diameter and originates from electron and x-ray scattering t h a t contributes to the analysis to a certain extent. The dark margin (c) m a y be due to condensation of material caused b y the beam and b y secondary excitation effects. Fig. 1 shows also the effects of moving the specimen under the static beam along a line of traverse to achieve a line scan (LS). To w h a t extent the secondarily excited area contributes to the analysis, thereby reducing resolution, is not known. The major portion of the excited x-ray spectra appears to originate from the area which is irradiated directly b y the beam. The secondarily excited area, however, grows with increasing section thickness since the electron beam has a
Electron Probe Analysis for Ion Transport Studies
347
high penetration power in soft biological tissue (Andersen, 1967; HShling and Hall, 1969). Embedded tissue exhibits a density of slightly above 1 g m1-1 (Hall and H6hling, 1969). One can estimate from Fig. 4 of H6hling and Hall (1969) that the depth of beam penetration is about 6 ~zin embedded tissue at 15 KeV accelerating voltage. The electron beam penetrates the 2 ~z sections completely. Hence, resolution is a function of section thickness. The depth of beam penetration can be decreased by working at low accelerating voltages, e.g. 5 KeV. The critical excitation potential of the K(Kc~) line is 3.606 KeV (Kemp). In order to achieve a high detection sensitivity, the accelerating voltage should be several times the critical excitation potential. Consequently, the use of an accelerating voltage lower than 15 KeV is not advised. In earlier studies involving the eryostat technique (L/~uchli, 1967a; l~asmussen et al., 1968), sections 16-20 ~ thick were routinely used at 25 KeV accelerating voltage. Assuming a density of 0.6 for such unembedded material, the depth of penetration of the beam is about 25 (HShling and Hall, 1969) and the resolution much poorer than for thin sections of epoxy resin embedded tissue. Sawhney and ZeIiteh (1969) conducted K determinations on freeze-dried portions of epidermal strips of leaves at 20 KeV. The depth of beam penetration is estimated to be 16 ~. The utilization of thin sections would increase resolution and also overcome some of the difficulties encountered with epidermal strips which may not have a smooth surface. Fig. 2 shows a line scan of K(Kz
Electron Probe Analysis of Freeze-Substituted, Epoxy Resin Embedded Tissue for Ion Transport Studies in Plants ANDR]~ L h v c n ~ , A~THU~ R. SPUR~ and RAr~OND W. WITTKOPP Departments of Soils and Plant Nutrition, of Vegetable Crops, and of Geology, University of California, Davis Received September 21, 1970
Summary. A new technique is described to prepare plant material for electron probe analysis. Root segments 1 mm in length were frozen at --170 ~ freeze-substituted with anhydrous ether at --30 ~ and infiltrated with Spurr's low-viscosity epoxy resin embedding medium at low temperatures. Sections 1 and 2 V thick were cut anhydrously using hexylene glycol in the ultramicrotome trough, mounted on the polished surface of a Be disc and vacuum coated with 150-200/~ aluminum. The new technique allows retention of water-soluble ions at the original sites in the tissue and is superior to eryostat sectioning in spatial resolution of electron probe analysis and in the preservation of cellular structures. The lateral transport of K + into the xylem of corn roots has been successfully studied by electron probe analysis of freeze-substituted, epoxy resin embedded material. Introduction Correlation of physiological experiments on ion transport in plants wiht structural investigations has recently gained much attention. Electron probe analysis and microautoradiography are two methods which yield direct results for localizing soluble ions transported in the plant, and mineral nutrients bound to the tissue. That these two methods supplement each other has been demonstrated in an experimental study (Li~uehli and Liittge, 1968). Mineral nutrients in the plant occur in part as solutes. During the preparation of specimens for electron probe analysis or microautoradiography, it is essential to minimize the displacement of inorganic solutes in the tissue. The techniques applicable to microautoradiography of soluble ions have been summarized by Lfittge and Weigl (1965). The eryostat technique is believed to allow ions to be retained at the original sites. Hence, it has been employed in the past to prepare plant material, for electron probe analysis (L~uehli, 1967a; Li~uehli and Ltittge, 1968; Rasmussen et al., 1968). However, cryostat sectioning of plant material does not yield thin sections and the preservation of cellular details is
342
A. L~uchli, A. R. Spurr, and R. W. Wittkopp:
poor. These are limiting factors i n achieving good spatial resolution in electron probe analysis. I n a n account of the factors t h a t determine resolution, A n d e r s e n (1967) states t h a t resolution depends on the diameter of the electron b e a m a n d on the v o l u m e of the specimen c o n t r i b u t i n g to the analysis. The volume of the sample excited is a f u n c t i o n of the depth of electron p e n e t r a t i o n a n d of electron a n d x - r a y scattering. Consequently, t h i n sections on the order of 1 ~ thickness are preferable to the 16-20 ~z sections o b t a i n e d b y the cryostat technique. I n this paper we report a new t e c h n i q u e to prepare sections of freezes u b s t i t u t e d material e m b e d d e d i n epoxy resin for s t u d y i n g ion t r a n s p o r t in p l a n t s t h r o u g h electron probe analysis.
Materials and Methods Plant Material. Seedlings of corn, Zea mays L., DeKalb 805, were grown as described by Lguchli and Epstein (1970). The seedlings were germinated in the dark for 4 days and then grown for 4 days in a growth chamber on 1/10 concentration Johnson nutrient solution (Johnson et al., 1957) but with Fe-EDTA supplied at 4/10 concentration. For the experiments, the root of an intact seedling was exposed for 3 hr at 30 ~ to an aerated solution of 0.2 mM KCI+0.5 mM CaSO4 and then excised just below the seed. A length of polyethylene tubing was attached to the root, and the root then immersed in an identical solution and allowed to exude for 1 hr. Thereafter, the root was rinsed with deionized water for 1 min and processed for electron probe analysis. Under these conditions, the translocation of K + and C1into the xylem exudate occurs at a steady rate (cf. L~,uehli and Epstein, 1970). 9 $'reezing and Freeze-Substitution. In a coldroom at 4 ~ root segments 1 mm in length were cut 10-11 mm proximal to the root apex and immediately frozen in a mixture of 8% (v/v) methyleyclohexane in isopentane cooled to --170 ~ with liquid nitrogen. The frozen tissue was dehydrated by freeze-substitution with anhydrous ether. Liittge and Weigl (1965) used ether in freeze-substitution to prepare specimens for microautoradiography and obtained results comparable to those using tissue dehydrated by freeze-drying or sectioned with a cryostat (Liittge and Weigl, 1965; Krichbaum et al., 1967). Prior to use, the ether was dehydrated with aluminum oxide (A1203, basic; Brockman, activity grade I) according to Wohlleben (1955). Four replicate root segments were transferred from the freezing mixture to dry ice and then plunged into a 25 ml polyethylene serew-cap vial containing 15 ml anhydrous ether + 2 g A1203, basic, at --30 ~ The vials were placed in a shaking device, which was operated in a low-temperature cabinet at --30 ~ and were continuously shaken for 7 days, at which time freeze-substitution was completed. Infiltration with Epoxy t~esin, and Embedding. Spurr's low-viscosity epoxy resin embedding medium E (Spurr, 1969) was chosen for infiltration and embedding. Based on the physical characteristics of medium E and its components shown in Table 1, the epoxy resin portion (vinylcyelohexene dioxide) was employed for initial infiltration into the tissue at --30 ~ the temperature used in freeze-substitution. Final infiltration was accomplished with the complete medium E starting at --12 ~ and finishing at + 4 ~. 1 Temperatures throughout the paper are in degrees centigrade (~
Electron Probe Analysis for Ion Transport Studies
343
Table 1. Physical characteristics o] Spurr's low-viscosity epoxy resin embedding
medium E and its components at low temperatures solid; -k fluid, high viscosity; -k -k fluid, medium viscosity; q- ~--k fluid, low viscosity. -
-
Temperature
Epoxy resina
Flexibilizer b
Anhydride Accelehardener c rater a
+++ §247247 §247247 §247247
+ §247 §247 §247
--§ §247
l~[edium E
(~ --30 --20 --12 + 4
+++ §247247 §247 §247
§ §247 §247247
a Vinyl cyclohexene dioxide (ERL-4206). b Diglycidyl ether of polypropylene glycol (D.E.R 736). e Nonenyl succinie anhydride (NSA). d Dimethylamincthanol (S-l). e 7.8 cP at 20 ~ ~ 60 cP at 25 ~
The freeze-substituted tissue segments were washed for 3 hr at --30 ~ with acetone dehydrated with Al203 (acid; Brockman, activity grade I) according to Wohlleben (1955). The acetone was changed every hour. Infiltration was started by adding to the last change of acetone an equal quantity of vinyl cyclohexene dioxide at --30 ~ After 4 hr the mixture was poured and drained from the vials and replaced by 100% vinylcyclohexene dioxide. Infiltration continued at --30 ~ overnight and then another change was made of the epoxy resin component before warming the vials up slowly to --12 ~ An equal quantity of the complete medium E was then added and allowed to stand until the evening. Another equal quantity of medium E was added and the vials were brought to -b4 ~ overnight. The tissue segments were then transferred to small vials containing 100% medium E and warmed to room temperature. After one additional change of the medium the specimens were transferred to oven-dried gelatin capsules containing medium E and cured for 16 hr at 70 ~ Specimen Preparation ]or Electron Probe Analysis. Polished discs of aluminum and beryllium, 2.54 cm in diameter, were used as section holders. The A1 discs were prepared essentially as described by L~uchli (1967a). Discs of pure Be with one side polished were obtained commercially (American Beryllium Corp., Inc., Sarasota, Florida). Transverse root sections 1 and 2 ~z thick were cut with a diamond knife on a Cambridge ultramicrotome. To avoid exposure of the sections to water (c/. Pease, 1966) anhydrous sectioning was accomplished through the use of hexylene glycol in the trough. The sections were removed from the surface of the glycol with a fine brush and placed on discs carrying a thin film of hexylene glycol. The discs were then placed in an oven at 70 ~ for 2 hr to spread the sections and to evaporate the hexylene glycol. When A1 discs were used ethylene glycol was employed as the fluid in the trough. J u s t prior to their use the surface of the A1 discs was pretreated with concentrated H~SO~ for about 10 rain, rinsed with deionized water, and dried in the oven to provide a surface which could be wetted with ethylene glycol. Otherwise
344
A. L/~uchli, A. R. Spurr, and R. W. Wittkopp:
the procedure was as described for Be discs. The discs with sections adhering were vacuum coated with 150-200 ~ A1. In this application, coating with A1 was found to be superior to carbon coating in that less damage was induced by the electron beam, probably because of better heat conduction. Elemental Analyses. Elements were measured with an Applied Research Laboratories electron probe x-ray analyzer, model EMX-SM, with scanning capabilities. The instrument was operated at 15 KeV accelerating voltage, 0.5 ~ electron beam diameter, and a sample current of 0.023 ~A on Be which was equivalent to 0.027 ~A on A1. Transmission Electron Microscopy. Electron micrographs from thin sections of freeze-substituted, epoxy resin embedded tissue were obtained by a slightly altered procedure. Root segments were freeze-substituted with ether as described, washed with acetone at --30 ~ and then fixed with 1% Os04 in acetone at --70 ~ overnight. At this temperature OsOa in acetone is stable (Feder and Sidman, 1958). The fixed material was washed with acetone, warmed to --30 ~ and infiltrated and embedded as described. Standard thin-sectioning procedures and contrasting with uranyl acetate and lead citrate were used. Electron micrographs obtained by this procedure will be published elsewhere (Li~uchli, Spurt and Epstein, in preparation).
Results and Discussion Measurement of b a c k g r o u n d r a d i a t i o n of Be a n d A1 are presented in Table 2. F o r analysis of K(K~) a n d Ca(K~), b a c k g r o u n d r a d i a t i o n of Be Table 2. Background radiation o/Be and A1 discs (counts per 100 sec) a
Be A1
K (Ks) (ADP)
Mg (Ks) (RAP)
Ca (Ks) (LiF)
1547 ~: 56.5 5283 • 102
944 • 26 37404 • 140.3
108 -4- 15 332 4-16
a Discs were vacuum coated with 150-200 A A1; electron probe x-ray analyzer was operated at 15 KeV with sample current of 0.023 ~A on Be which is equivalent to 0.027 ~A on Al; analyzing crystals are given below the elements analyzed (the recently introduced crystal RAP is rubidium dihydrogen phosphate); the data represent the means of 4 replicates with standard errors.
is only a b o u t 30% of t h a t of A1. The A1 we used contains Mg as a n i m p u r i t y a n d is therefore i n a d e q u a t e for the m e a s u r e m e n t of Mg i n tissue sections. Beryllium is the superior s u p p o r t i n g m a t e r i a l because a low b a c k g r o u n d r a d i a t i o n is needed to increase the peak to b a c k g r o u n d ratio in electron probe analysis. Table 3 shows d a t a on b a c k g r o u n d r a d i a t i o n of epoxy resin on Be w i t h o u t p l a n t tissue present. B a c k g r o u n d readings of K ( K ~ ) r a d i a t i o n from sections of polymerized epoxy resin on Be a n d from Be alone are n o t significantly different. However, the s t a n d a r d error of the m e a n of b a c k g r o u n d m e a s u r e m e n t s rises with a n increasing thickness of resin. The
Electron Probe Analysis for Ion Transport Studies
345
Table 3. Background radiation o/Be disc and o/epoxy resin sections (without tissue) on Be discs (counts per 100 sec)a
Be Epoxy resin (1 ~) Epoxy resin (2 ~)
K (Ks) (ADP)
Mg (Ks) (RAP)
Ca (Ks) (LiF)
C1 (Kc0 (ADP)
1551 ~ 66.9 1808:t:113 1725___254
986 :t: 55 979~ 14 986~ 165
108 • 10 101:J-10 118•
312 • 7.8 3082-[-439 5696•
a Samples were vacuum coated with 150-200A A1; electron probe x-ray analyzer was operated at 15 KeV with 0.023 ~*Asample current; analyzing crystals are given below the elements analyzed; the data represent the means of 4 replicates with standard errors.
resin is not contaminated with Mg and Ca, but it contains appreciable amounts of C1 which causes a high standard error for the background detection of CI(K~) radiation in 2 bt thick sections. Similar values of background radiation were obtained when the readings were taken in the resin-impregnated tissue and in the resinous embedding material outside of the tissue. Epoxy resins based on epichlorohydrin may eontMn small amounts of C1. Ingram and I-Iogben (1968) report that Epon 826 contains 30 millimoles kg -1 of organically bound C1. Some C1 also occurs in the ionic form in resins of this type. Vinyl cyelohexene dioxide, the principal epoxy resin employed in our study, is not derived from Cl-eontMning compounds and is presumed to have very low levels of C1. The epoxy resin flexibilizer D.E.t~. 736 in our embedding mixture is derived in part from epiehlorohydrin and averages 0.5% C1. ttowever, D.E.IK 736 is only a small part of the mixture and on the basis of the total resinous formulation the C1 content is about 0.073%. Still, this level is too high for studies in which C1 is of interest. Yet, it is possible to prepare epoxy resins with very low levels of C1. Another alternative is to consider embedding in methaerylate (Gielink et al., 1966), but GMIe and Stuve (1968) do not recommend it because of its relative instability under the electron beam. Methaerylate also has other disadvantages sueh as marked shrinkage and bubble formation during polymerization. Our preparation technique was applied to a study on the lateral transport of K + into the xylem of corn roots (Liiuehli. Spurr and Epstein, in preparation). Preliminary experiments showed that the electron probe x-ray analyzer was utilized at the limits of its detection sensitivity when K was measured in sections 1 bt thick. More aceurate data were obtained with 2 b~ sections in evaluating the pattern of K distribution in transverse sections of roots. Further, the electron beam caused less noticeable damage in 2 bt sections as compared with thicker sections. 23 ]?lant~(Berl.), ]~d.95
346
A. Lguchli, A. P~. Spurr, and R. W. Wittkopp:
Fig. I. Transverse section, 2 ~ thick, of a corn root taken 10-11 mm proximal to the root apex. Root segment frozen at --170 ~ freeze-substituted with anhych'ous ether and embedded in low-viscosity epoxy resin medium. Section mounted on a Be disc, cotaed with 150-200 A A1 and photographed by reflected-light microscopy. Section shows the effects of point analysis (PA) with central dark spot (a), secondarily excited area (b) and dark margin (c), and also the effects of a line scan (LS). Endodermis (E), pericyele (P), xylem vessel (Xy). • 720
Fig. l shows part of a transverse section, 2 ~ thick, taken 10-11 m m proximal to the root apex. The effects of exposing the section to a static electron beam for point analysis (PA) are visible at several sites. The central dark spot (a) is caused directly b y the beam and indicates the p r i m a r y spatial resolution which is better t h a n 1 ~z. There is also a secondarily excited area (b) surrounded b y a dark margin (c). The area (b) is about 7 ~z in diameter and originates from electron and x-ray scattering t h a t contributes to the analysis to a certain extent. The dark margin (c) m a y be due to condensation of material caused b y the beam and b y secondary excitation effects. Fig. 1 shows also the effects of moving the specimen under the static beam along a line of traverse to achieve a line scan (LS). To w h a t extent the secondarily excited area contributes to the analysis, thereby reducing resolution, is not known. The major portion of the excited x-ray spectra appears to originate from the area which is irradiated directly b y the beam. The secondarily excited area, however, grows with increasing section thickness since the electron beam has a
Electron Probe Analysis for Ion Transport Studies
347
high penetration power in soft biological tissue (Andersen, 1967; HShling and Hall, 1969). Embedded tissue exhibits a density of slightly above 1 g m1-1 (Hall and H6hling, 1969). One can estimate from Fig. 4 of H6hling and Hall (1969) that the depth of beam penetration is about 6 ~zin embedded tissue at 15 KeV accelerating voltage. The electron beam penetrates the 2 ~z sections completely. Hence, resolution is a function of section thickness. The depth of beam penetration can be decreased by working at low accelerating voltages, e.g. 5 KeV. The critical excitation potential of the K(Kc~) line is 3.606 KeV (Kemp). In order to achieve a high detection sensitivity, the accelerating voltage should be several times the critical excitation potential. Consequently, the use of an accelerating voltage lower than 15 KeV is not advised. In earlier studies involving the eryostat technique (L/~uchli, 1967a; l~asmussen et al., 1968), sections 16-20 ~ thick were routinely used at 25 KeV accelerating voltage. Assuming a density of 0.6 for such unembedded material, the depth of penetration of the beam is about 25 (HShling and Hall, 1969) and the resolution much poorer than for thin sections of epoxy resin embedded tissue. Sawhney and ZeIiteh (1969) conducted K determinations on freeze-dried portions of epidermal strips of leaves at 20 KeV. The depth of beam penetration is estimated to be 16 ~. The utilization of thin sections would increase resolution and also overcome some of the difficulties encountered with epidermal strips which may not have a smooth surface. Fig. 2 shows a line scan of K(Kz
