On the preparation of cryosections for immunocytochemistry

JOURNAL OF ULTRASTRUCTURE RESEARCH

89, 65-78 (1984)

On the Preparation of Cryosections for Immunocytochemistry GARETH GRIFFITHS, ALASDAIR M c D O W A L L , R U T H BACK, AND JACQUES DUBOCHET

European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Republic of Germany Received May 18, 1984, and in revised form August 1, 1984 The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 Din.

© 1984 A c a d e m i c Press, Inc.

tration of sucrose. In the subsequent steps of the procedure, the section is thawed and the reaction with antibodies is made in a liquid medium. The Tokuyasu cryosection technique, like all postembedding immunocytochemical methods, offers the advantage that access of antibodies to every cellular compartment, at least on the surface of sections, is assured. It is also the method which gives the best preservation of antigenic determinants, since the only potential denaturation step before labeling is the initial aldehyde fixation. When the thawed cryosectioning technique itself was introduced, excellent fine structure preservation from a range of tissues was demonstrated on stained, air-dried sections (Tokuyasu, 1973). The negative staining procedures used at that time helped protect sections against air-drying artifacts. The high degree of contrast provided by such staining, however, made it incompatible with antibody labeling studies. The immunomarkers ferritin, and to a lesser extent, colloidal gold (which was introduced for this technique by Slot and Geuze, 1981) were difficult, if not impossible to visualize. Initially, a compromise was reached using low-

Immunocytochemistry using t~ozen thin sections has become an important technique in cell biology during the last few years, following the pioneering work of Tokuyasu (1973, 1978) and Tokuyasu and Singer (1976). It has allowed a number of interesting and novel observations to be made especially in the study of membranes (see Gfiffiths et al., 1983a,b, and Slot and Geuze, 1983, for references), as well as of the cytoskeleton and muscle (Geiger et al., 1981; Tokuyasu et al., 1981, 1983; Chen and Singer, 1982). The technique has, however, not yet gained a widespread usage, notably because of difficulties encountered in structural preservation by several users. The aim of the current study is to provide a better understanding of the method in order that greater control of its critical steps is made possible. For this purpose we have used a spherical tissue culture cell, Chinese hamster ovary (CHO), as a model system. The essential steps in the Tokuyasu technique are shown in Fig. 1. In this method, the purpose of the freezing step is simply to make a hard block of tissue that facilitates sectioning. To reduce freezing damage, the tissue is impregnated with a high concen65

0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

GRIFFITHS ET AL. ALDEHYDE FIXATION

1982, 1983a,b). This procedure is in itself not fully understood, nor is it always sat0.6-2.3 M SUCROSE INFUSION isfactory, leading to continual attempts at (CRYOPROTECTANT) i m p r o v e m e n t s (e.g., Tokuyasu, 1980a; Grif(Tokuyasu, 1973; Geuze and Slot, 1980) fiths et aL, 1982; see below). In particular, l COOLING IN LIQUID NITROGEN even in our best thin sections, holes up to 1 1 # m in diameter are f o r m e d in some parts CRYOSECTIONING o f the cytoplasm. Examples are shown in i SECTION RETRIEVED AND THAWED ON A Fig. 2. These holes are mostly air-drying artifacts and this will be demonstrated in LOOP OF 2.1-2.3 M SUCROSE AND TRANSFERRED ONTO this paper. GRIDS The direct observation o f frozen hydrated l sections in the cryoelectron microscope has GRIDS LABELED WITH ANTIBODY recently b e c o m e a practical m e t h o d (Dul bochet et aL, 1983; McDowall et aL, 1983). PROTHN-A--GOLD (Slot and Geuze, 1981) OR FERRITIN-CONJUGATED SECOND It involves observing, at low temperature, ANTIBODY cryosections o f the vitrified native sample. (Tokuyasu and Singer, 1976) In this way one overcomes the usual prepl aration artifacts except those due to the secCONTRAST WITH URANYL ACETATE tioning process itself. The study o f this l AIR-DRY IN A FILM--METHYL CELLULOSE m e t h o d has given us a better understanding (Tokuyasu, 1978; Griffiths et aL, 1982) (OR A MIX- o f the freezing, as well as o f the cutting proTURE OF METHYL CELLULOSE (+ POLYETHcesses (Chang et aL, 1983). For example, it YLENEGLYCOL) AND URANYL ACETATE (Tohas shown that vitrification, i.e., solidifikuyasu, 1980b) cation without ice-crystal formation, is posFIG. 1. The Tokuyasu thawed frozen section tech- sible and is indeed a prerequisite for avoidnique. ing freezing d a m a g e . In a d d i t i o n , the possibility o f quantitative mass thickness determination on frozen hydrated sections er concentrations o f stain which was far from provides a powerful tool for characterizing satisfactory, because first, structures could the observed material. For example, the not be clearly visualized and second, air- comparison o f the same section, before and drying artifacts became a serious problem after freeze-drying, gives an estimate o f its (Tokuyasu, 1980b). water content. The introduction o f methyl cellulose to The goal o f the present work is to learn protect thawed frozen sections against sur- m o r e a b o u t the T o k u y a s u c r y o s e c t i o n face tension damage caused by air-drying m e t h o d by studying the intermediate prepwas an essential step in the d e v e l o p m e n t o f aration steps with the hydrated cryosection the technique (Tokuyasu, 1978). This subtle and mass m e a s u r e m e n t methods. In particprocedure determines, together with the ini- ular we have addressed the following questial aldehyde fixation, the structural pres- tions: ervation as well as the contrast o f the sec1. W h a t is the state o f water in the altions. The latter can vary from positive dehyde-fixed, sucrose-infused frozen samcontrast (Tokuyasu, 1978) where m e m - ple? Further, is the fixed cell permeable to branes appear dense on a lighter back- sucrose? ground, to partially negative contrast where 2. What is the appearance o f sections o f m e m b r a n e s are negatively contrasted but the spherical cells in the frozen hydrated other cell structures, such as the nucleus, form; how do these compare with sections show positive contrast (see Griffiths et aL, that have been thawed and dried, with or

CRYOSECTION PREPARATION

without methyl cellulose? Further, how do they compare with Epon sections? 3. What is the average thickness of the frozen hydrated sections? Does this thickness change upon thawing and drying? Again, what is the influence of methyl cellulose on the thickness of the dried section? MATERIAL AND METHODS

Cellpreparation. Chinese hamster ovary (CHO) culture cells were grown in spinner culture (i.e., suspension). The cells were cultured on a minimal essential medium containing 10% fetal calf serum and 5% CO2. In a few cases, baby hamster kidney (BHK) cells were infected with Semliki Forest virus (SFV). The culturing of these cells, infection with SFV and labeling with antibodies against the spike proteins of the virus was carried out as described previously (Green et aL, 1981; Grifliths et al., 1982, 1983a,b). An aliquot o f the ClIO culture medium was taken and centrifuged for 2 rain at 4000g to form a pellet which was immediately fixed. The cell pellet was washed briefly with phosphate-buffered saline and then fixed at room temperature with 1% glutaraldehyde in 100 m M P i p e s buffer, pH 7.0, with 5% sucrose (wt/wt). The pellet was resuspended in this solution and after 30 rain centrifuged at 13 000g for 1 rain. The pellet was then washed with 100 m M P i p e s with 10% sucrose and could be stored, when necessary, for up to 4 weeks at 4 ~ in the presence of 0.02% (wt/wt) sodium azide. Tokuyasu method. The procedures we use for performing the various steps in Fig. 1 have been described in detail in two recent publications (Griffiths et al., 1983b; Griffiths, 1984). We would like to add some points here and emphasize others. 1. A 2.1 M sucrose solution is used here instead o f a 2.3 M solution simply because it is slighly less viscous and allows easier manipulations of the small pieces (0.5-1 m m 3) of cell pellet prior to freezing. Infusion is allowed to continue for 15-30 rain, before freezing in liquid nitrogen. 2. Cryosectioning for the Tokuyasu procedure was made at -110°C in a Sorvall MT2B ultramicrotome equipped with an FC-2 cryochamber. 3. Sections are cut with glass knives prepared by the Tokuyasu procedure (Griffiths et aL, 1983a,b), which are then coated with tungsten, exactly as described by Roberts (1975). Conditions must be worked out empirically so that a very light shadow is made which gives a light-grey tint on a filter paper strip. Too much tungsten causes the section to stick to the glass. This procedure has the advantage that sections can be cut more smoothly than using uncoated knives and that the knives can be stored longer than uncoated knives. Furthermore, our best knives can be used for several hours and may even be reused on other days (up to

67

four different times). They are simply rinsed with distilled water and dried with Freon gas after use. 4. Care was taken in this study to ensure that the same range of section thicknesses were made as are used for our routine antibody labeling studies. This is determined on the knife edge in two ways. First, by the "fluidity" of the sections as they are cut: thinner sections are more fluid or "cellophane-like" whereas the thicker sections come off as rigid sheets. Second, by interference colors, which are less reliable because, unlike sections floating on a liquid, the sections are not all lying in one plane. Our sections range from gold through purple to blue. 5. For section thickness measurements, the fold method of Small (1968) was used (see Griftiths et aL, 1983b, for illustration). During the procedure for collecting and thawing the cryosections using the sucrose loop in the cryochamber, the best sections are always obtained when the sections contact the sucrose before it has solidified. When the latter occurs, extensive folding of the sections on the grid is observed, as a result of the sections not being allowed to stretch on the sucrose drop. These folds are, to a large extent, retained as the drop is further thawed at room temperature. The folds are protected from air-drying collapse by the methyl cellulose in the same way that cell structures are protected. The folds can therefore be used in the same way to estimate thickness as for Epon sections (Weibel, 1979); in the case of Epon, extensive folds appear when sections are picked up on grids from above, as opposed to below, the water level. 6. We now routinely use newborn calf serum (GIBCO) instead o f gelatin in order to reduce background labeling. This serum which contains only traces oflgG, which do not bind protein A, was found empirically to reduce background significantly during immunoblotting experiments at EMBL. It also gives minimum background labeling on thawed frozen sections, especially when labeling with very sticky proteins, such as lectins or some monoclonal antibodies. The grids are treated for 10 rain with a 10% solution of this serum in phosphate-buffered saline before the antibody reactions and all antibody and protein-A--gold solutions are made with this mixture (stock solutions of which are stored at -20°C) (see Griffiths et aL, 1983a,b). 7. We are now routinely using a new drying procedure which is a variation of Tokuyasu's adsorption staining method (Tokuyasu, 1980b). The sections are postembedded in a relatively high concentration of lowviscosity methyl cellulose. The aim here is to impregnate as much o f the section as possible by using a smaller-molecular-weightcompound. Specifically, a 2o/0 Methocell (25 cP; Fhika AG) solution is made (instead of the Tylose MH, 300 cP, used previously) by mixing the powder with water at 95"C, then putting the mixture immediately on ice, and then further stirring at 0-4"C for about 24 hr. The solution can be kept at this temperature for at least 3 more days. It is centrifuged at 55 000 rpm with a Beckman 60Ti or 70Ti rotor for 1

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GRIFFITHS ET AL.

hr. The tubes are stored in the refrigerator without disturbing the debris that precipitate. Small aliquots from the surface of this solution are taken immediately before use and mixed with a stock 3°/0solution ofuranyl acetate to give a final concentration of 0.1-0.4% uranyl acetate. Grids are floated on this solution, on ice for 10 min, then looped and excess fluid removed with filter paper and dried quickly in a chamber containing silica gel. Care should be taken that only enough fluid should be removed to give interference colors from gold to blue after drying. With increasing stain concentration there is more tendency for negative staining. In our experience, good contrast and preservation are more reproducible with this method than with earlier procedures. Thawed and air-dried cryosections. The same preparation procedure as for the Tokuyasu cryosections was used but the unstained sections were simply air-dried after the final water washing (after removing excess water with filter paper) instead of applying the methyl cellulose solution. Hydrated cryosections. Hydrated cryosections were prepared from the fixed, sucrose-infiltrated frozen sample according to the method described previously (McDowall et al., 1983). Most cryosections were obtained on the SorvaU cryoultramicrotome at -110°C. Some sections were obtained at - 160°C with glass or diamond knives in a Reichert RC4 cryoultramicrotome. In every case, they were transferred with an eyelash from the knife to the supporting grids, pressed between two cold metal pieces and transferred into the cryoelectron microscope (Philips 400 equipped with the cold stage PW 6591 / 100) where they were observed around -160°C with minimal electron exposure. The state of the ice was determined by electron diffraction (Dubochet et aL, 1982b). Epon sections. A pellet of fixed cells was prepared and embedded according to a standard Epon embedding procedure, consisting of a mixture of Epon 812, methyl nadic anhydride, dodecenyl succinic acid anhydride, and 2,4,6-Tris(dimethylaminomethyl)phenol (Grittiths et aL, 1983a). Sections were obtained using a diamond knife either by the normal ultramicrotomy method, in which they are floated on water before being picked-up on the grid (referred to below as: standard Epon) or by dry cutting at room temperature in which the sections were cut on a dry knife, transferred to the grid with an eye-lash, and flattened by pressing them between two metal blocks in a way similar to the one used for hydrated cryosections (referred to below as:

dry Epon). Dimensions and density. The dimensions of the ceils were measured in suspension with an optical microscope and on electron micrographs of the sections. In the latter case the width of D of the cell measured perpendicularly to the cutting direction was determined as also the width d parallel to it. The percentage compression was then estimated as (D - d)/d x 100.

The magnification of both light and electron microscopes was calibrated with a grating replica. The mass thickness, pt (i.e., the product of the density, 0 by the thickness, t), of unstained sections was determined from contrast measurements on the micrographs. The method has been described elsewhere (Eusemann et aL, 1982). The geometry used in this work leads to a mean free mass thickness of 230 mg m -2, (see Eusemann et aL, 1982). The degree of hydration, defined as the ratio of the amount of water leaving the section upon freeze-drying in the microscope to the total mass of the hydrated frozen sections was determined from the comparison of the mass thickness before and after freeze-drying, after correcting for the shrinkage which takes place during the process (Chang et al., 1983). The thickness of the hydrated cryosections was deduced from the mass thickness as well as from the hypotheses that the extracellular 2.1 M sucrose solution has a density of 1.18 g - cm-3 and that this would be reduced by 7% due to vitrification (Dubochet et aL, 1982a). Furthermore, we assumed that the extracellular regions (which contained sucrose only) had the same section thickness as the adjacent intracellular regions. For the air-dried sections, the thickness was estimated in two ways. First, from the length of the shadow around the edge of the section, the shadowing angle (ca. 24 °) being determined from the length of the shadow of polystyrene spheres. Second, by a new method, to be described elsewhere (Berriman et al., 1984) in which the thickness is calculated using a trigonometric method from a series of tilted micrographs of sections coated with gold particles. RESULTS

General Aspect of the Tokuyasu Dried Frozen Sections The Tokuyasu cryosection method gives a good representation of the cell and this has been abundantly illustrated before (see r e f e r e n c e s i n t h e i n t r o d u c t i o n ) . F i g u r e 2 illustrates the possibilities as well as limitations of the method. Many cell structures are well preserved and contrasted. However, the asterisks in this figure illustrate obvious structural artifacts. These artifacts are partly the result of stretching of the sections during thawing, but are primarily due to surface-tension damage resulting from airdrying that occurs, despite the presence of methyl cellulose-stain mixture. Some of these artifacts are the result of deformations of existing structures such as the lysosome

CRYOSECTION PREPARATION in Fig. 2. Others are clearly f o r m e d de novo. I f a thinner methyl cellulose layer is used, the contrast increases but the n u m b e r o f artifacts also increase significantly (Griffiths et al., 1983b). This is best illustrated in Figs. 3 and 4 where C H O cells are air-dried in the complete absence o f stain or methyl cellulose (there is no h e a v y metal present). At low magnification (Fig. 3) cell contours are maintained (there was no significant difference in cell profile diameters from the methyl cellulose dried cells; results not shown) and very good contrast is observed. At higher m a g n i f i c a t i o n (Fig. 4), h o w e v e r , severe structural damage is obvious, especially the large holes, m o s t o f which are probably derived from small vacuoles, the network appearance o f the nucleus and cytoplasm, and the complete collapse o f microvilli at the surface.

Frozen Hydrated Sections W h e n sections from the same block used for the T o k u y a s u procedure were observed in the frozen h y d r a t e d state, a n u m b e r of striking differences in the physical characteristics o f the sections were evident. The first is the low contrast o f the frozen hydrated cells. This is because the density o f the extracellular sucrose solution is close to that o f the biological material. The c o n t o u r

69

o f the cell can, however, be identified une q u i v o c a l l y by b e a m - i n d u c e d b u b b l i n g (Dubochet et al., 1982a) or when the sections are freeze-dried by rewarming above ca. - 9 0 ° C . The second difference is the presence o f severe cutting artifacts. As shown in Fig. 5 knife marks, crevasses and distortions are obvious; these are characteristic artifacts in frozen hydrated sections (Chang et al., 1983). They disappear in the thawed, methyl ceUulose-protected cryosection (Fig. 6). Further, cells which appear spherical by light microscopy (see below) are oval in shape in the hydrated cryosection, due to section compression. U p o n thawing, on a drop o f concentrated sucrose solution, the cells regain their r o u n d profile (Fig. 6). This p h e n o m e n o n o f compression u p o n cutting and reexpansion on a liquid surface is not inherent to cryosectioning since the same effect is observed with Epon sections (Figs. 7 and 8). Quantitative data on these effects are shown in Table I.

Sphericity o f the Cells C H O cells appear spherical by light microscopy: they have an average diameter o f 13.9 _ 2.3 #m. U p o n glutaraldehyde fixation they shrink Slightly and b e c o m e more i r r e g u l a r in o u t l i n e ( a v e r a g e d i a m e t e r : 11.5 _+ 2.5 /xm). W h e n the fixed cells are

FIG. 2. An example of a thawed Tokuyasu frozen section selected to show the air-drying problem. The section, of baby hamster kidney cells infected with Semliki Forest virus, has been labeled with antibodies to the spike (coat) proteins of the virus followed by protein-A--colloidalgold. Specific labeling of the membranes of the Golgi stack (G) and of structures characteristically seen in infected cells, referred to as capsid structures (arrowheads). (For more information on this system, see Griffiths et aL, 1982, 1983a,b.) Whereas the fine structure preservation and contrast in general is acceptable, the asterisks indicate structural artifacts in the Golgi area (top right) free in the ctyoplasm and in a lysosomal structure (L). While these may be due, in part, to stretching of the sections, upon thawing, most are due to air-drying artifacts. Bar = 100 nm. x 57 000. FIG. 3. Sections of CHO cells prepared by the Tokuyasu procedure except that they were air-dried without uranyl acetate or stain. The cell dimensions are the same as with the normal procedure with methyl cellulose. Note the very high contrast, even in the complete absence of heavy metal. Large "vacuoles" are evident in the cytoplasm and the cell in the upper left corner of the micrograph is torn. Bar = 1 #m. × 2500. FIG. 4. Higher-magnificationmicrograph of the same grid as in Fig. 5 to show the severe air-drying artifacts, especially the large holes (asterisks) which appear as vacuoles at lower magnification. Also evident is a network appearance of the nucleus and cytoplasm and a collapse of microvilli at the cell surface (arrow). Bar = 1 ~zm. x 16 000.

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G R I F F I T H S ET AL.

';

~ S ¸"

E

CRYOSECTION PREPARATION

71

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GRIFFITHS ET AL.

73

CRYOSECTION P R E P A R A T I O N TABLE I ESTIMATION OF COMPRESSION Treatment 1. 2. 3. 4. 5.

Tokuyasu frozen sections Air-dried frozen sections Frozen hydrated sections Normal Epon sections Dry Epon sections

N u m b e r of ceils measured 140 50 150 30

Ratio 0.99 1.05 0.58 1.05 0.66

D/d ~

% Compression

_+ 0.2 +_ 0.18 + 0.12 +_ 0.28 +_ 0.1

0 0 42 _+ 11 0 34 +_ 12

a Where D is the cell diameter in the direction of cutting and d is the diameter perpendicular to it.

rinsed briefly in Pipes buffer with 10% sucrose and then infused with 2.1 M sucrose they swell to an average diameter approaching that of the unfixed cell (14.7 +_ 1.7 ~tm). Electron microscopic measurements of the average diameter of the cell profiles on sections prepared either with the Tokuyasu procedure or with Epon give approximately the same result in agreement with the optical microscopy measurements. The size and shape of the fixed cell is therefore well preserved during these preparation procedures. Vitrification

Hydrated cryosections of cells infiltrated with 2.1 M sucrose are completely vitrified. They appear glassy and section well at - 100 to -160°C. When the sections are freezedried by warming them in the electron microscope the e v a p o r a t i o n process ends around - 9 0 ° C before ice crystallization takes place. When the cells were infused with 1 M sucrose, the extracellular space was in the form of hexagonal ice crystals, whereas the water inside the cells was vitrified or was crystallized into the unusual cubic ice form. Further, the blocks appeared more milky than our routine blocks infused with 2.1 M sucrose and they sectioned poorly.

When 0.6 M sucrose is used to infuse, hexagonal ice crystals were usually found both inside and outside the cells and the blocks were even more difficult to section. Section Thickness

Table II summarizes the results of our mass thickness m e a s u r e m e n t s of both frozen hydrated and air-dried (without methyl cellulose or stain) sections as well as direct thickness m e a s u r e m e n t s of the sections prepared by the Tokuyasu procedure and of the air-dried sections. For the former, we believe the thickness determined by the fold procedure is underestimating the real thickness, since, in general, the most convincing folds are seen on sections in the thinner range. For the latter, the unidirectional shadowing with platinum gave a mean thickness of 50 + 18 nm. This value is probably an overestimate because of the rim frequently formed around the cells during drying. The result using the quantitative stereology method, which we believe to be more reliable, is the one given in Table II. DISCUSSION

The central importance of the sucrose for the success of the Tokuyasu method is underlined by the results presented here.

FIG. 5. Hydrated cryosection of a CHO cell infused with 2,1 M sucrose. The cell contours are indicated by arrows and severe compression is evident. The arrowhead shows the direction of cutting, which is evident from the n u m e r o u s knife marks. Bar = 1 gin. x 12 000. Fie. 6. Cryosection from the same block as the section from Fig. 3 which has been prepared by the Tokuyasu procedure. Notice the absence of knife marks, as well as compression and the good contrast obtained with the uranyl acetate-methyl cellulose mixture. Bar = 1 ~m. x 10 500.

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G R I F F I T H S E T AL.

m

FIG. 7. Epon section o f a C H O cell cut on a dry knife (arrowhead indicates knife marks). Note the severe c o m p r e s s i o n in the cutting direction. Contrasted with o s m i u m tetroxide, uranyl acetate, a n d lead citrate. C o m p a r e with Fig. 3. Bar = 1 gin. × 6700. FI~. 8. Similar to Fig. 7 except that the sections were floated on water before being picked up on grids. Bar = 1 /~m. x 10 0 0 0 .

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CRYOSECTION PREPARATION T A B L E II ESTIMATION OF SECTION AND MASS THICKNESS

Treatment

Section Section thickN u m b e r thickness ness of uncorcorsections retted rected c used (nm) (nm)

Mass thickness uncorreeted (rag m -2)

Mass thickness corrected C C o m p o n e n t s (nag m -2) measured Tissue, sucrose, water

Frozen h y d r a t e d sections (intracellular)

52

109 a

238 +_ 43

136

Frozen h y d r a t e d sections (extracellular)

52

109 a

227 + 41

130

Sucrose, water

180

86

Tissue, sucrose

30.5

Tissue

Freeze-dried sections T h a w e d , washed, air-dried frozen sections

56

31

31 b

Dried frozen sections (Tokuyasu)

55

71 +__ 25

30.5 +_ 9.5

71 b N o t m e a s u r e d due to presence o f m e t h y l cellulose

a O b t a i n e d by calculation f r o m m a s s - t h i c k n e s s m e a s u r e m e n t s . b O b t a i n e d by direct m e a s u r e m e n t . c Corrected for c o m p r e s s i o n a n d / o r shrinkage.

First o f all, we confirm the expectation f r o m previous works (see Carstenson et al., 1971; Pentilla et aL, 1974) that the sucrose solution penetrates fixed cells. This fact is demonstrated from mass thickness measurements (Table II): on the one hand, the average intracellular mass thickness in frozen hydrated sections was found to be 136 mg m -2 and this value decreases to 86 mg m -2 u p o n freeze-drying. The contribution o f water in the frozen hydrated section is therefore 50 mg m -2. On the other hand, the washed, air-dried sections were found to have an average intracellular mass thickness o f 30 mg m -2, which, c o m p a r e d to the 86 mg m -z o f the freeze-dried, sucrose-conmining sections lead to the estimation that the contribution o f sugar in the frozen hydrated section is 56 mg m -2. The ratio o f sucrose to water inside the cell is therefore 56:50 which corresponds to a 1.9 M solution o f sugar. This is in agreement with the value o f 2.1 M for the sucrose solution actually used for infiltration. The second major observation is that fixed cells infused with 2.1 M sucrose are vitrified when cooled as described in this work. It has been shown recently (Adrian et aL, 1984;

McDowall et aL, 1984) that freezing damage is essentially nonexistent in vitrified material. Consequently, no i m p r o v e m e n t in the preservation o f the sample can be expected by using more elaborate cooling methods. Caution should be taken when dealing with lower sucrose concentration or with other tissues. In the original 1973 publication, Tokuyasu r e c o m m e n d e d sucrose in the range 0.6-1.6 M depending on the tissue. The precise concentration was empirically determined by reference to the sectioning process and to the final fine structure p r e s e r v a t i o n o b t a i n e d o n the n e g a t i v e stained, i.e., dried sections. The need for different sucrose concentrations is perhaps a reflection o f the different water content o f the various tissues. I f 2.1-2.3 M sucrose cannot be used for any reason, it would seem logical to use more efficient coolants, such as Freon or liquid propane. This has been pointed out before (Singer et al., 1982). As indicated previously (Dubochet, 1982b), an ultimate assessment o f the state o f the water in the section is only possible by electron diffraction o f the hydrated section. It happens, however, that hexagonal ice which forms when freezing is not optimal is m u c h

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more difficult to cut so that an experienced cryomicrotomist can decide already on the microtome if a specimen is correctly frozen. Another fortunate consequence of the high sucrose concentration is to increase the devitrification temperature: whereas pure vitreous water crystallizes around - 135°C, water in the concentrated sucrose starts to crystallize at much higher temperature. For this reason, cryosectioning of vitrified samples is still possible up to - 8 0 to -70°C. In the original publication (Tokuyasu, 1973), sucrose was one of many different compounds used as cryoprotectant. As discussed in detail by Tokuyasu (1973, 1978, 1980a,b), the role of these low-molecularweight compounds is, however, not only that o f cryoprotection; their presence influences the plasticity and therefore cutting properties o f the blocks. High sucrose concentrations, for example, make the blocks relatively soft. This was the reason why Tokuyasu (1973) did not recommend such high concentrations, at the cutting temperatures then used ( - 70 to - 90°C). At - 110°C the high sucrose concentration is ideal for cutting sections in the 50- to 110-nm range but makes it more difficult to cut very thin (<50 nm) sections. Using lower temperatures may improve matters but, when very thin sections (20-30 rim) are required, as obtained by Tokuyasu (1974), lower sucrose concentrations and therefore more efficient coolants would have to be used. On this point, it is important to stress that in this present study our interest is to describe the properties o f average sections that are easily and routinely obtained rather than the thinnest ones. The observation of frozen hydrated sections and o f dry Epon sections shows that the cutting process causes more damage than is generally believed. Noteworthy in particular is the severe compression along the cutting direction as well as the crevasses. Most o f these artifacts are, however, suppressed when the section is floated on the liquid. It is the good fortune of the electron micros-

copist that they remained unnoticed with standard procedures. The treatment with methyl cellulose used here is, in general, an adequate method to p rot ect biological structures during the drying process. Whereas the original average thickness of our frozen sections is 110 nm, they are after drying estimated to be 70 rim. As pointed out the latter estimate is surely an underestimation and the real thickness after drying is surely closer to the thickness of the frozen hydrated section. Nevertheless, some collapse does occur, as illustrated in Fig. 2. Some structures, such as membrane vacuoles are clearly more susceptible to damage. The collapse phenomenon becomes severe where the sections are air-dried, without methyl cellulose or uranyl acetate, and the thickness is reduced to about 30 nm. In this case, the density of the material reaches the high value of 1 g cm 3. The positive staining method of Tokuyasu (1980a) whereby low concentrations of methyl cellulose (0.2%) and uranyl acetate (0.01-0.1%) are mixed with a high concentration (2%) of low-molecular-weight polyethylene glycol should, in principle, reduce if not eliminate the air-drying collapse phenomenon. Indeed, with this technique the artifacts we have discussed here are far less frequent. Another factor, which must now be taken into consideration, however, is contrast. The contrasting method we use is, a subtle mixture o f positive and negative contrast depending on the structure involved. In the case of membranes, which provide the main focus of our interest in immunolabeling studies, the strong negative contrast facilitates identification of membrane profiles, even when obliquely sectioned. Immunogold particles, but not ferritin, are still easily visualized. In the case of the positive staining procedure only membrane profiles which are transversely sectioned will be visualized. This makes very thin (< 100 nm) sections a prerequisite. We believe that, at the present time, our contrasting method is the

CRYOSECTION PREPARATION

most practical compromise for visualizing membranes and gold particles on the one hand, and providing protection against airdrying on the other. Our hydrated cryosections are thicker than our methyl cellulose dried sections. In other words the real thickness of the sections is greater than we had previously thought and certainly thicker than routine Epon sections. Great caution must therefore be exercised in interpreting labeling of small structures. For example, quantitative determination of antigens inside vesicles requires to take into account those vesicles which are completely within the section and are thus not penetrable by the label. The exact knowledge of the real section thickness is a parameter which influences such determination. In our opinion, the most fundamental limitation we are left with in the Tokuyasu technique is the chemical crosslinking by the fixative which seems unavoidable for any immunocytochemical technique at the electron microscopical level. In spite of an enormous amount of literature, the structural and immunological effects of fixation remain poorly understood because this treatment is generally associated with osmication, dehydration, infiltration, and polymerization of the plastic. The improved understanding o f the Tokuyasu method gained through the present work, and the possibility of comparing fixed with unfixed tissue using frozen hydrated sections, open greater possibilities for exploring how this treatment influences the morphology of biological structures. We thank Dr. Frank BooN and Dr. Jean-Claude Jesior for their advice and critical comments. The thickness measurements of the air-dried frozen sections were made by John Berriman. We also acknowledge the suggestions made on the methyl-cellulose technique by Dro Alan McKenzie.

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