Histochemistry54, 39- 50 (1977)
Histochemistry 9 by Springer-Verlag 1977
Phenidone-Ascorbic Acid Development in Electronmicroscopic Autoradiography Harry Heijnen and Hans Geuze Centre for Electron Microscopy,Schoolof Medicine,State Universityof Utrecht, Nicolaas Beetsstraat22, Utrecht, The Netherlands
Summary. Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible. Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.
Introduction Many photographic developers are used for electronmicroscopic autoradiography (EMA), and have been tested with respect to their resolution, efficiency and other parameters (Salpeter et al., 1969; Vrensen, 1970; Wisse and Tates, 1968; Kopriwa, 1967b, 1975). The most commonly used developers are Kodak Microdol-x (Caro and Van Tubergen, 1962); Kodak D 19 b (Kopriwa, 1967b); Gold latensification/ Elon ascorbic (Wisse and Tates, 1968); p-phenylenediamine (Caro and Van Tubergen, 1962). Lettr6 and Paweletz (1966) first described phenidone-ascorbic acid as a useful developer and since then it has been used in several EMA studies (Berg and Young, 1971; Young, 1973; Basinger et al., 1976; Slot et
40
H. Heijnen and H. Geuze
al., 1974; Slavkin et al., 1976; Slot et al., 1976; Markov et al., 1976; Kramer and Geuze, 1977) Autoradiographic parameters, however, have never been investigated for phenidone-ascorbic acid. The aim of this study is to calibrate this developer in combination with Ilford L4 as photographic emulsion. Microdol-x developer was used as a reference. We determined resolution and efficiency, and investigated the spatial reiationship between developed silver grains and the silver bromide crystals from which they originated.
Materials and Methods Autoradiographical Procedure For EMA, sections of the radioactive sources (see below) were transferred to collodion-coated glass slides and covered with a thin layer of carbon. The slides were covered with a slightly overlapping layer of silver bromide crystals of Ilford L4 emulsion, using a dipping apparatus as designed by Kopriwa (1967a). The emulsion was diluted 1:2,5 with demineralized water. The uniformity of the emulsion layer was checked in the electronmicroscope. Exposure took place in light tight boxes at 4 ~ C. Exposure times varied from 1 to 143 days. Development was in phenidone-ascorbic acid, prepared according to Lettr6 and Paweletz (1966), for 1 min at 16~ C, and in Kodak Microdol-x for 5 rain at 19~ 1 We fixed in 20% Na-thiosulphate, and rinsed three times in demineralized water. Collodion film and sections were floated onto water. Grids placed over the sections and film plus grids were removed from the water by means of a lens paper and allowed to d r y . Then the grids with sections were carefully removed and viewed in a Zeiss EM9A electronmicroscope. The precise magnification of each micrograph taken was calculated with the aid of a replica grid.
Radioactive Sources Plane source. For determination of grain yield and efficiency we used a uniformly labeled 3H-methylmethacrylate sheet (initial specific activity 10 mci/g, Radiochemical Centre, Amersham, G.B.). The actual specific activity was measured as follows. Samples of the labeled methacrylate sheet were weighed and dissolved in 10 ml dioxane scintillation fluid. Desintegrations per minute (dpm) per unit volume were counted in a Packard scintillation counter. At the time when the methacrylate was used, the specific activity was 6.5 mci/g. Total decays per square micron were then calculated from the actual specific activity and the specific density of methacrylate (1.17 g/cm3). The radation dose was 2.5 decays per gm 2 per day, calculated for a 0.1 ~tm (pale goldish) thick section. The efficiency of the techniques for each exposure time was calculated from: grains/~tm 2 decays/lain a
x 100%.
Line source. A radioactive source of 3H-albumin was prepared as follows. Non-radioactive albumin was labeled with tritium by means of reductive methylation (Means, 1968). Triated NaBH 4 (+1 rag; specific activity 8.5 mci/mg) was added to a 2 ml albumin solution (7 mg/ml) at pH 9.0 and 0~ Then a 3.7% formaldehyde solution was added slowly during 30 rain (5 times 10 Id). After labeling, free NaBH4 was removed from the albumin by dialysis against a 3.3 m M phosphate buffered 1% NaC1 solution (pH 7.2) during 2 days. The final concentration o f the ~H-albumin solution was about 8 mg/ml and the specific activity was 40 mci/g. The radioactive albumin solution was pipetted onto glass slides and allowed to dry vertically. 1 Gold latensification may precede phenidone and microdol-x development, thus improving efficiency (Gupta et al., 1973)
Development in EM Autoradiography
41
The albumin layer was fixed with paraformaldehyde vapour for 5 min. After fixation the albumin layer was divided into squares with a razor blade, and stripped off onto water with the aid of hydro fluoric acid. The squares were picked up on prepolymerized epon blocks. After complete drying a second layer of epon was added, thus forming a sandwich of radioactive albumin between two layers of epon. After polymerization, sections with a pale gold interference colour were cut at right angles to the albumin film and treated as described in the autoradiographical procedure. The profile of the cross-sectioned albumin film in the autoradiographs was considered as a line source. The thickness of the line was approximately 25 nm.
Resolution Enough line was photographed to yield more than 500 developed silver grains per developer. The distance of the midpoint of each silver grain to the centre of the line was measured at both sides of the line up to a distance of 2tam. In the case of microdol-x the centre of the smallest circular circumference of the grain was taken as the midpoint of the grain. For both developers a resolution value was obtained from a cumulative grain distribution, as previously described by Salpeter and Bachmann (1969). They defined the Half Distance (HD) as the distance to a line source, which includes 50% of the grains. Background was determined in areas at more than 2 gm away from the line.
Relation BetweenGrains and Crystals Al:15 diluted Ilford L14 emulsion was applied to collodion coated slides as described above. The slides were placed in a vacuum evaporator and coated with a platinumcarbon layer during 45 sec. The angle and distance between the slides and the evaporation source were respectively 30~ and 8 cm. The emulsion was exposured to photons, which were emitted during the evaporation process. After shadowing, the emulsion was developed in phenidone or microdol-x, fixed, rinsed, and mounted on grids. When viewed in the EM, both the shadow of the crystals and the developed silver grains were visible.
ResuLts Appearance o f the Silver Grains T h e t w o d e v e l o p e r s u s e d in this s t u d y p r o d u c e d d i f f e r e n t l y s h a p e d silver g r a i n s (Figs. 3 a n d 4). P h e n i d o n e p r o d u c e d small, r o u n d e d o r h e x a g o n a l grains, w h e r e a s m i c r o d o l - x r e v e a l e d l o n g f i l a m e n t o u s silver grains. A f t e r p h e n i d o n e d e v e l o p m e n t , t h e g r a i n s o f t e n d i f f e r in size f r o m o n e d e v e l o p m e n t to a n o t h e r . A t h i g h e r d e n s i t i e s p h e n i d o n e g r a i n s w e r e o f t e r g r o u p e d in c l u s t e r s o r s h o r t r o w s , b u t r e m a i n e d d i s t i n c t a b l e . F i g u r e 1 gives t h e d i m e n s i o n s o f b o t h t y p e s o f silver grains. T h e v a l u e s r e p r e s e n t m e a s u r e m e n t s o f one d e v e l o p m e n t . T h e r e m a y be s o m e v a r i a t i o n s b e t w e e n d i f f e r e n t e x p e r i m e n t s , w h i c h w e r e m o r e i m p o r t a n t f o r p h e n i d o n e . T h e f i g u r e s h o w s t h a t t h e a v e r a g e d i a m e t e r o f t h e silver g r a i n s is 140 n m f o r p h e n i d o n e a n d 360 n m f o r m i c r o d o l - x .
Grain Yield and Efficiency T h e yield o f a u t o r a d i o g r a p h i c g r a i n s s t r o n g l y d e p e n d s o n t h e d e v e l o p e r used. F i g u r e 2 gives t h e i n c r e a s e o f g r a i n d e n s i t y o v e r 3 H - m e t h a c r y l a t e s e c t i o n s w i t h
42
H. Heijnen and H. Geuze
9 phenidone [ ] microdol- X
2O
100
200
Fig. 1. Frequency histogram of grain sizes after phenidone and microdol-x development. With grain size is meant the diameter of the phenidone silver grain and of the smallest circumference of the microdol-x grain. For each developer a total of 80 grains was measured
300 400 500 grain size (nm)
Q
|
25
~2o o--o phenidone i--&
O ///I /
~E ~ 20
microdol - X 0
15
150 / f ~/
10,
phenidone
O
1'0
20
3[]
4'0
Exposure
5'0 time
(days)
--11
40
60
8[]
100
120
1,~0
Exposure t i m e ( d a y s )
Fig. 2 a and b. Regression lines of grain density against exposure time. a Short exposure times were 1 , 2 , 4 , 6 , 8 , 16, 22, 36, 44 days. o oy=0,21 x+0,37 r=0.98; zx-A y=0,11 x + 0 , 1 4 r = 0 , 9 8 . b Long exposure times were 33, 44, 88, 143 days. o - o y = 0 , 1 1 x+5,73 r=0,97. Only phenidone developed autoradiographs could be counted because of the distinctness of the grains
increasing exposure times. Both developers show a significant linear relationship for the short exposure times (P < 0.005). In the case of microdol-x, grain counting was impossible at exposure times longer than 36 days, because the filamentous silver grains began to intermingle. Phenidone remained countable even up to 143 days (Fig. 2b). Then the increase of the grain density remained linear (P<0.025). Figure 2a already indicates that phenidone is more sensitive than microdol-x. The background for both developers remained within 4% of the grain yield.
Development in EM Autoradiography
43
Table 1. Per cent efficiency calculated for each exposure time by dividing total number of grains per gm 2 with total decays per /,tin2. Radiation dose was 2.5 decays per gm 2 per day (radiation dose is the total decays per lama per exposure time, including the 50% emitted away from the emulsion) Exposure-time Radiation dose (days) (decays/Bm 2)
% Efficiency (_+ SD) phenidone
l 2 4 6 8 16 22 36 44 88 143
2.5 5 l0 15 20 40 55 90 110 220 357
17.2• 23.4• 13.8 15.2_+ 6.2 8,6 • 10,6_+ 7.1 _+ 8.7_+ 10,2_+ 7.3 + 6.2_+
3.6 3.0 3.0 1.8 1.2 0.9 0.9
microdol-x
8.2• 6.2• 4.1• 3.6• 5.5• 3.8• 4.2•
Figs, 3 and 4. Autoradiographs of pale gold cross-sections through radioactive albumin film embedded in epon ( x 18,500). Fig. 3. developed with phenidone Fig. 4. developed with microdol-x
44
H. Heijnen and H. Geuze
80
microdol - X
60
)Ltl
40
20
.. =,,~JKA Ul
.E E
01
80
"6 JCl
E
3 Z
phenidone
60
40
1700 1360 10'20 680
3~
0
340
680 1020 1360 1700
DislQnce
(nm)
Fig. 5. Histogram showing the grain distribution at both sides of a thin tritium labeled albumin line source (hatched), and for both sides together (white). Each column represents a distance of 43 nm. At 0 is the position of the line source
Development in EM Autoradiography
45
Efficiency values were calculated for all exposure times and expressed as percentages of the radiation dose. Table 1 shows that on an average the efficiency of phenidone is twice that of microdol-x at all exposure times. Initially efficiency drops with increasing radiation dose. This is of about the same order for both developers. After 6 or 8 days the efficiency becomes more or less constant and the SD's decrease with increasing exposure. The SD's of the efficiency values reflect variations of the grain yield. These variations are higher for the long exposure times, because then less samples are taken in order to count about the same number of grains.
Resolution Figures 3 and 4 show the distribution of silver grains at both sides of the radioactive line source. For H D measurement we used autoradiographs with low grain densities to facilitate accurate measuring. The distances between grains and line were tabulated in a frequency histogram with distance steps of 43 nm (Fig. 5). Since the distribution of the grains at both sides of the line (o in Fig. 5) appeared statistically normal, the distances were replotted in a one-sided form (Fig. 5, white columns). Figure 6 gives the cumulative presentations of
10 0.9
0.8 0.7
r
06
-
'6
phenidone
............ m i c r o d o [
-X
O.5 o 04 ~6
,o
0.3
]l HD
P Q2 la.
0.1
340
680
1020
1360
1700
2040 Distance (nrn)
Fig. 6. Cumulative histograms of the unilateral!y plotted histograms in Figure 5. Each histogram column gives the fraction of silver grains that layed within a certain distance from the line source. The half distances (HD) can easily be read from these histograms (arrows)
Fig. 7. Ilford L4 emulsion, 1:2,5 diluted, after shadowing with platinumcarbon and development in phenidone. Both, silver grains and greyish remnants of the shadowed crystals are visible. Occasionally two or more silver grains seem to originate from one crystal (arrows). Most of the grains remain within the boundary of the crystal ( x 22,500) Figs. 8 and 9. Ilford L4 emulsion, 1:15 diluted, after shadowing and development (x22,500). The greyish remnants of the crystals are widely dispersed, permitting a better location of the individual silver grains. Fig. 8. developed with phenidone. Note the small size of the silver grains, possibly representing the sites of the latent images. All grains fell within the perimeters of the parent crystals. Fig. 9. developed with microdol-x. The microdol-x grains grow beyond the bounderies of the crystals into the direction of the shadow cone. The other sides of the crystals are presumably shielded by the platinumcarbon layer
Development in EM Autoradiography
47
the unilateral plotted histograms of Figure 5. The distance, within which 50% of the total grains fell (HD), can be read from these cumulative curves. The HD values were 175 nm for phenidone and 130 nm for microdol-x. Background over the resolution specimens was 0.02 grains/gin 2 for phenidone and 0.002 grains/btm 2 for microdol-x.
Relation Between Grains and Crystals Figure 7 shows a mono layer of Ilford L4 emulsion crystals after shadowing and development in phenidone. The greyish globules represent remnants of the shadowed silver bromide crystals. Rounded silver deposits lie mostly within these remnants. Because of the tightly packed crystals no evidence could be obtained with respect to the number of grains per crystal and the exact location of the grains. For these reasons we carried out the same shadowing experiment a 1:15 diluted emulsion. In such a '~spaced ''-emulsion the silver deposits after phenidone development were very small 2, and nearly all were located within the perimeter of the crystal from which they originated (Fig. 8). Over 80% of the 400 crystals counted produced only one deposit. Development with microdol-x resulted in silver grains with about normal shape and size (Fig. 9). The microdol-x grains always remained in contact with the silver bromide crystals, but in contrast to phenidone, often grew out of their perimeter. Because of the coiled shape of the microdol:x grains it was not possible to determine the exact number of grains per crystal.
Discussion
Dimensions and shape of the silver grains after 1 min development with phenidone at 16~ correspond to those observed by Kopriwa (1975) (solution A in her study). Within one development, variations in grain size were larger for microdol-x than for phenidone. On the other hand variations from one development to another were ofter found to be larger for phenidone, possibly due to variations in processing which is more critical for phenidone. Phenidone developer has to be used within 1 h after preparation. In contrast microdol-x can be preserved for at least one month. The shadow technique has proved to be valuable in investigating the relationship between developed silver grains and their parent crystals. The tiny silver deposits in the shadowed autoradiographs of diluted emulsion layers, developed with phenidone, presumably represent the sites of the latent images of the crystals. It appeared that each crystal produced one silver grain. As compared with normal autoradiographs, the silver grains in the shadowed autoradiographs were smaller, especially when phenidone was used. It therefore seems possible that the carbonplatinum layer had a shielding effect on the silver bromide z A 1:15 diluted emulsion, developed with a solution physical developer (as phenidone) may produce smaller grains, because the total amount of silver ions, needed for reduction into metallic silver grains is reduced
48
H. Heijnenand H. Geuze
crystals. Short development (as with phenidone) might then produce very small grains. Accordingly, autoradiographs of a layer of diluted emulsion revealed smaller grains than those of a monolayer (Fig. 8). In unshadowed normal autoradiographs the diameter of the phenidone grains (140 nm) were within the range of the parent crystals. Phenidone grains never seem to unite into larger silver conglomerates. Taken together, our observations indicate that each excited crystal may reveal one silver grain after phenidone development. Weber (1969) described that phenidone development in combination with Ilford L4 revealed silver grains that were smaller and were more tightly packed than the original silver bromide crystals. From a non-quantitative evaluation of autoradiographs Weber (1969) suggests that several latent images may be formed within one silver bromide crystal, thus giving rise to more grains per crystal. This is not in agreement with our findings that most silver bromide crystals form just one silver deposit (possibly representing the site of the latent image). It should be emphasized that the shadowed autoradiographs were exposed to photons, which were emitted during the shadowing process. Though not likely, the relationship between grain and crystal may be different after irradiation with electrons. Experiments are underway in which normal exposed (i.e. with electrons) emulsion are shadowed after development, thus avoiding the effect of the platinumcarbon on the developing process. The linear increase of the number of grains with increasing exposure times suggests that there is no significant latent image fading or emulsion saturation for a period up to 140 days. The data presented in Table 1 were obtained by dividing the functions of grains/gm ~ versus exposure time and decays/gm2 versus exposure time. The first function was determined by means of a regression analysis, revealing regression lines (y=ax+b), which intersected the abcis at negligible distances b, possibly caused by a low initial background. For the short exposure times the constant factor b will contribute more to the result of the ratio, than for long exposure times. The initial drop of efficiency for both developers is probably caused by this phenomenon. Despite individual variations, the average efficiency becomes constant within 10 days exposure. As radioactive source for resolution determination we have chosen a fixed, proteinaceous source, comparable to the sources in labeled biological material. The HD value for microdol-x (130 nm), obtained with this source, is slightly better than the 160 nm found by Salpeter et al. (1969). The difference may be caused by the nature of the sources used. The HD value for phenidone (175 nm) in combination with Ilford L4 is not statistically higher than that for microdol-x. (Wilcoxon, two sample test, c~=0.01). Thus the smaller photographic error in the case ofphenidone does not significantly improve resolution (Bachmann et al., 1968). The resolution of phenidone is within the range of many other developer-emulsion combinations, and can be considered as acceptable for quantitative use. The background fog was considerable higher for phenidone than for microdol-x in all experiments. However, this background is negligible low compared to the much higher grain densities in labeled biological material. For instance,
DeveIopment in EM Autoradiography
49
autoradiographs of pancreatic tissue, developed with phenidone, showed a background, which was within 1% of the grain densities of the labeled material (Slot et al., 1976).
Conclusions
Using Ilford L4 as photographic emulsion, phenidone-ascorbic acid is about twice as sensitive as microdol-x, and its resolution is slightly lower than that of microdol-x. The HD value of phenidone however, is within the range of many other developers. For quantitative EM autoradiography phenidone seems an extremely useful developer, because the silver grains remain distinct, even at high grain densities.
References Bachmann, L., Salpeter, M.M., Salpeter, E.E. : Das Aufl6sungsverm6gen elektronenmikroskopischer Autoradiographien. Histochemie 15, 234-250 (1968) Basinger, S., Bok, D., Hail, M.: Rhodopsin in the rod outer segment plasma membrane. J. Cell Biol. 69, 29 42 (1976) Berg, N.B., Young, R.W.: Sulfate metabolism in pancreatic acinar cells. J. Cell Biol. 50, 469 483 (1971) Caro, L.G., Van Tubergen, R.P.: High resolution autoradiography. I. Methods. J. Cell Biol. 15, 173-188 (1962) Gupta, B.L., Moreton, R.B., Cooper, N.C.: Reconsideration of resolution in EM autoradiography using a biological line source. Microscopy 99, 1-25 (1973) Kopriwa, B.M. : A semiautomatic instrument for the radioautographic coating technique. J. Histochem. Cytochem. 14, 923-928 (1967a) Kopriwa, B.M.: The influence of development on the number and appearance of silver grains in electron microscopic radioautography. J. Histochem. Cytochem. 14, 501-515 (1967b) Kopriwa, B.M.: A comparison of various procedures for fine grain development in electron microscopic radioautography. Histochemistry 44, 201 224 (1975) Kramer, M.F., Geuze, J.J. : Glycoprotein transport in the surface mucous cells of the rat stomach. J. Cell Biol. 73, in press (1977) Lettr6, H., Paweletz, N. : Probleme der elektronenmikroskopischen Autoradiographie. Naturwissenschaften 53, 268 271 (1966) Markov, D., Rambourg, A., Droz, B.: Smooth endoplasmic reticulum and fast axonal transport of glycoproteins, an electron microscope radioautographic study of thick sections after heavy metal impregnation. J. Microsc. Biol. Cell. 25, 57-60 (1976) Means, G.E., Feeney, R.E.: Reductive alkytation of amino groups in proteins. Biochemisry 7, 2192-2202 (1968) Salpeter, M.M., Bachmann, L., Salpeter, E.E. : Resolution in electron microscopic radioautography. J. CelI Biol. 41, 1 20 (1969) Slavkin, H.C., Mino, W., Bringas, P. Jr.: The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration. Anat. Rec. 185, 289 312 (1976) Slot, J.W., Geuze, J.J., Poort, C. : Synthesis and intracellular transport of proteins in exocrine pancreas of frog (Rana Esculenta). I. An ultrastructural and autoradiographic study. Cell Tissue Res. 155, 135-154 (1974) II_ An in vitro study of the transport process and the influence of temperature. Cell Tissue Res.167, 14%t65 (1976) Vrensen, G.F.J.M. : Some new aspects of efficiency of electron microscopic autoradiography with tritium. J. Histochem. Cytochem. 18, 278 290 (1970)
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Weber, H.: Einige Erfahrungen bei der Anwendung der etektronenmikroskopischen Autoradiographie. Acta. biol. med. Germ. 22, 159-167 (1969) Wisse, E., Tates, A.D. : A gold latensification- Elon ascorbic acid developer for Ilford L4 emulsion. Fourth European Regional Conference on Electron Microscopy. Rome, p. 465-466 (1968) Young, R.W.: The role of the golgi complex in sulfate metabolism. J. Cell Biol. 57, 175-189 (1973)
Received June 7, 1977
