CELL ORGANELLES AT UNCOATED CRYOFRACTURED SURFACES AS VIEWED WITH THE SCANNING ELECTRON MICROSCOPE

Transcription

1 J. Cell Sci. 21, (1976) 47 Printed in Great Britain CELL ORGANELLES AT UNCOATED CRYOFRACTURED SURFACES AS VIEWED WITH THE SCANNING ELECTRON MICROSCOPE P. S. WOODS AND MYRON C. LEDBETTER Biology Department, Queens College, Flushing, New York 11367, U.S.A. and Biology Department, Brookhaven National Laboratory, Upton, New York 11973, U.S.A. SUMMARY A method of direct visualization of cell organelles by scanning electron microscopy (SEM) is described. Plant and animal tissues fixed in glutaraldehyde and osmium tetroxide are treated with the ligand thiocarbohydrazide and a second osmium tetroxide solution, to increase their osmium content. Tissues are then dehydrated, infiltrated with an epoxy monomer, and together solidified with dry ice and fractured. The pieces are transferred to pure acetone, critical-point dried, attached to stubs with silver paint and viewed by SEM. The ligating procedure increases the osmium concentration at its original bonding site sufficiently to render the tissue electrically conductive, thus obviating the need for metallic coating. The organelles at the fractured surface are revealed in relation to their osmium incorporation rather than by surface irregularities as with coating methods. The image derived from the uncoated surface approaches in resolution that of transmission electron micrographs of thin sections. A portion of the image arising from a small distance below the surface, while at progressively lower resolution, provides some 3-dimensional information about cell fine structure. INTRODUCTION Observation of plant or animal cell organelles by scanning electron microscopy (SEM) based on surface irregularities has been demonstrated by others using various methods (Guttman & Styskal, 1971; Lim, 1971; Panessa & Gennaro, 1972, 1973; Humphreys & Wodzicki, 1972; Tanaka & lino, 1972; Humphreys, Spurlock & Johnson, 1974). In one method, Tanaka & lino (1972) exposed the contents of cells by cracking frozen, osmium-fixed tissues infiltrated with monomeric epoxy resin. These workers identified some of the organelles by small surface irregularities in the otherwise smooth break after the pieces were dried and coated with a heavy metal. We repeated this method and found by careful stereoscopic observation of pairs of micrographs that some of the secondary electron signal was derived from below the coated surface, presumably from the osmium absorbed by particular structures during fixation. We sought ways to enhance that part of the image which comes from the osmium-rich organelles, while diminishing that part which shows surface irregularities. After several attempts we found a way to view the fractured surface directly without coating, thus permitting us to observe the organelles at and just beneath the

2 48 P. S. Woods and M. C. Ledbctter surface at relatively high resolution. This was accomplished by using the thiocarbohydrazide (TCH)-osmium ligating method developed by Seligman, Wasserkrug & Hanker (1966) to stain tissue sections for transmission electron microscopy (TEM), and later modified by Kelley, Dekker & Bluemink (1973) to render bulk tissues electrically conductive for surface viewing with the SEM. Using this procedure, it became apparent that osmium ligation in combination with resin cracking provides a new way to study cell fine structure with the SEM. We present here a more extensive account of our preliminary findings reported earlier (Woods & Ledbetter, 1974). MATERIALS AND METHODS Preparation of tissues prior to fixation We explored the usefulness of the method for plants and animals by using corn root tips and portions of tadpole tail. Corn (Zea mays L.) seeds were germinated in the dark at 18 C between layers of thick filter paper moistened with distilled water and grown for 8 or 9 days prior to fixation. Frog (Rana catesbeiana Shaw) tadpoles approximately 6 cm long were collected in the wild and maintained in the laboratory in 15-I. tanks under conditions similar to those in nature. A relaxed condition of the tail muscle was achieved prior to fixation by forcing the tadpole to succumb to exhaustion. Samples were taken from root tips and small pieces of tadpole tail (2 mm') containing epidermis and muscle. Buffers and washes The buffers for the fixing solutions and washes were 005 M throughout, with Sorenson's phosphate at ph 6-8 being used for the corn roots and cacodylate at ph 7-4 being used for the tadpole tails. Washes in buffer were done 3 times at i-h intervals, and washes in water were done 4 times at 05-h intervals. Washes after OsO 4 and TCH treatment were at 45 C C to ensure removal of free reagent and prevent the deposition of TCH crystals in the tissue. Preparation of the TCH The often pinkish TCH crystals (05 g in a small beaker) were washed by repeated rinsing with distilled water until colourless. The washed crystals in 25 ml of water were heated to 60 C C for a few minutes, and then cooled to room temperature for 1 h to attain saturation at that temperature. During preparation the solution was shielded from strong light and before use was filtered by syringe through a Millipore filter of o^y-fim pore size. Dehydration and infiltration of tissue Dehydration of samples was done in a graded series of acetone-water mixtures at concentrations of 10, 30, 50, 70, 90% and pure acetone, all carried out in an ice bath to minimize osmotic effects, with changes at 05-h intervals. The specimens in pure acetone were warmed to room temperature and 2 more changes of acetone were made to assure dryness. Infiltration with epoxy resin monomer (Epon 812) was through a series of 25, 50, and 75% monomer in acetone at 2-h intervals, followed by pure monomer overnight and daily changes of pure monomer for 3 more days using a tissue rotator set at 1 rev/min. Fracturing, drying and mounting of tissue on stubs Tissues to be cracked in epoxy monomer were positioned in a shallow well drilled in a 6-mm aluminium plate with about half of the tissue projecting from the well and with a minimum of resin left in the well. The plate with the tissue was chilled with powdered dry ice to solidify

3 Cell organelles viewed with SEM 49 the resin with the tissue. To do this it was found convenient to use an ice bucket with a tightly fitted lid to prevent ice crystals from forming on the tissue. Cracking was accomplished by a single thrust of a knife precooled with liquid nitrogen. Dry ice may be used to chill the knife if care is taken to keep the knife thoroughly cooled prior to making the fracture. The plate and all of the cracked pieces of tissue were plunged into acetone at room temperature. The cracked tissues were collected in vials, washed a few times with pure acetone to remove the resin and dried by the critical point method of Anderson (1951) using liquid CO 2. Acetone was the transition fluid (Porter, Kelley & Andrews, 1972). The dried pieces were attached to aluminium stubs with silver paint and examined uncoated by SEM at from 10 to 30 kev with the fracture face of the specimen oriented nearly normal to the electron beam. Outline of the procedure The excised tissues to be prepared for SEM were: (1) fixed in buffered glutaraldehyde (3 %) f r J h an d 6 % for 2 h, the latter finally heated to 45 C for 15 min for more complete fixation, cooled to room temperature and washed in buffer; (2) treated in 2% buffered OsO 4 overnight and washed in water at 45 C; (3) treated with saturated TCH at room temperature for 3 min, heated to 45 C for 15 min, washed with water at 45 C and cooled to room temperature; (4) treated with 1 % aqueous OsO, for 1 h and rinsed with water; (5) dehydrated in cold acetone; (6) infiltrated with epoxy resin monomer; (7) chilled, cracked, and washed in acetone; (8) dried by the critical point method; and (9) mounted on stubs and observed by SEM. For some specimens steps 3 and 4 were repeated (osmium ligated twice) to increase the osmium content even further. Procedure for TEM Samples for TEM were processed through steps 1 through 5 (above), infiltrated in an epoxy mixture based on Luft (1961), polymerized at 60 C C for 3 days, sectioned with a diamond knife, and observed without further staining at 80 kev. RESULTS Figs. 1 and 2 compare the results achieved by the two methods of rendering tissue electrically conductive. In Fig. 1 a xylem initial cell in interphase from a corn root tip was fractured and coated with gold according to the procedures of Tanaka & lino (1972). The limits of the cell are outlined by the wall, which appears bright due to the roughness of the fracture through the fibrous wall. Conspicuous vacuoles appear within the cytoplasm especially in the upper portion of the figure. A large nucleus is located centrally, with its lighter nucleolus and presumed heterochromatin bodies against the darker nucleoplasm. Presumably most of the secondary electrons detected here arise from the gold coat applied to reveal the surface and provide a conductive path for electrons; however, stereoscopic study of pairs of such images lead us to the conclusion that a portion of the signal, notably that from parts of the nucleolus and chromatin, originates from below the coat, probably from the denser concentrations of osmium present in these regions. The remainder of the cytoplasm shows some irregularities in the fracture plane induced by various organelles associated with such cells, but interpretation is difficult at best. In contrast, Fig. 2 shows a cell probably in interphase and is from a corn root tip rendered electrically conductive by osmium ligation. The root was fractured in the same manner as that used for the cell in Fig. 1, but in this case is viewed uncoated. In this method sufficient

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5 Cell organelles viewed with SEM 51 reduced osmium has accumulated in the specimen to provide a conductive path by which excess electrons may pass from the specimen to the grounded stub, thus avoiding charging effects. The image in Fig. 2 is derived almost exclusively from secondary electrons with little contribution from primary backscattered electrons as determined by the almost undetectable image displayed upon reversal of the V from the collector screen to 100 V to visualize backscattered electrons. The signal of secondary electrons will vary with the concentration of osmium within the tissue, rather than with surface irregularities, as for a metal-coated specimen. This difference in the way images are formed is illustrated by the appearance of the cell wall which has a relatively low affinity for osmium and as a consequence appears dark in Fig. 2 though bright in Fig. 1. The contents of the vacuoles appear similar with the 2 methods because of their particulate nature and osmium affinity; however, the limiting vacuolar membrane, or tonoplast, stands out in bright contrast in Fig. 2 because of the high osmium affinity relative to the surrounding cytoplasm, whereas such contrast is lacking in the metal-coated sample (Fig. 1), which shows the roughsurfaced cytoplasm simply terminating abruptly at the tonoplast. Membranes form a prominent feature of the cytoplasm in the uncoated fractured cell treated by the ligation method (Fig. 2), making it possible to identify such structures as the endoplasmic reticulum, nuclear envelope and mitochondria with cristae. These, as well as dictyosomes, and small vesicles making up the cell plate of cells in telophase have already been demonstrated (Woods & Ledbetter, 1974) in similarly treated corn roots. This is what one would expect from the distribution of osmium in cells as seen in thin section by TEM. Besides the highly visible membranes, the nucleolus and condensed heterochromatin bodies (barely visible in coated specimens) stand out in bold contrast against the darker nucleoplasm. The more diffuse euchromatin is also visible. In the coated specimen (Fig. 1) the image formed from secondary electrons reveals chiefly surface characteristics and shows little of the underlying osmium concentrations; however, in the uncoated, ligated sample, the secondary electron picture indicates osmium concentrations not only at the fracture surface, but also deeper within the specimen. This enhanced depth of imaging is illustrated in Figs. 3 and 4. Fig. 1. A scanning electron micrograph of a gold-coated xylem initial cell in interphase from a corn root tip fixed in buffered glutaraldehyde then OsOj and fractured in frozen epoxy resin monomer. Identifiable structures are: nucleus (n), nucleolus {mi), vacuoles (i>) and cell wall (cw). The structure marked he is presumed to be heterochromatin. Other organelles of the cytoplasm though detectable are of uncertain identity, x Fig. 2. A scanning electron micrograph of an uncoated meristematic cell probably in interphase from a corn root tip processed by the osmium ligation method but otherwise fixed and fractured as in Fig. 1. The richness of structure revealed in contrast to Fig. 1 is striking. The limits of the nucleus are clearly defined by its envelope (ne). Within the nucleoplasm the nucleolus (nu), condensed heterochromatin (lie) and diffuse euchromatin (ec) are seen in high contrast. Cytoplasmic organelles include: vacuoles (v), endoplasmic reticulum (er), and mitochondria (m) with cristae. x 8500.

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7 Cell organelles viewed with SEM 53 In Fig. 3 the fracture plane passes through a large filamentous mitochondrion in 3 places (arrows). That portions of the mitochondrion between breaks lie below the fractured surface is obvious from the micrograph of this uncoated and ligated specimen. From the predominant absence of condensed chromatin and general appearance of the nucleus the cell in Fig. 3 is judged to be in interphase. In Fig. 4 there are portions of 2 cells, the upper of which is in metaphase and shows various organelles distributed around the central zone of condensed chromosomes appearing as bright objects lying in a darker field. The nuclear envelope is lacking, as expected. Stereo views of pairs of micrographs of this cell give the impression that the chromosomes are rounded, with the less well defined and darker portions lying below the fractured surface. As is apparent in the micrograph, the microtubules of the spindle are not distinguishable. The web-like material that is visible in the spindle region (Fig. 4) is inconsistent with the image of microtubules usually obtained by TEM. In the lower cell of this illustration the nuclear envelope is clearly intact and, from the distribution of condensed chromatin, we estimate the cell to be in mid-prophase. Figs. 5 and 6 are of similarly treated cells from the epidermis of a frog tadpole tail as seen respectively in thin section by TEM and in fractured surface by SEM. Fixation and ligation of the 2 samples was identical and both are viewed without further metallic staining or coating. The identifiable structures in Fig. 5 include the nucleus with its double membrane, plasma membrane, endoplasmic reticulum, mitochondria, dense pigment granules, keratin filaments, desmosomes, basal lamina, orthogonal arrays of collagen and intercellular canaliculi. Most of the structures identified in Fig. 5 are also seen in Fig. 6, though with reversed contrast. The general location of the basal lamina with its associated filaments is visible as a bright zone adjacent to the collagen fibres. It is impossible to distinguish individual keratin filaments or ribosomes in this micrograph (Fig. 6); however, the outer and inner membranes of the nuclear envelope including pores (arrows of Fig. 6A) are easily visualized. Figs. 7 and 8 illustrate, respectively, a transmission micrograph and a scanning micrograph of striated muscle from frog tadpole tail. All of the bands and filaments typical of relaxed myofibrils seen in the transmission micrograph of Fig. 7 are also visible in the scanning micrograph of Fig. 8. These structures include the Z lines marking the limits of the sarcomere of each myofibril, the I bands with actin filaments Fig. 3. A scanning electron micrograph of portion of an uncoated cell in interphase from a corn root tip osmium ligated and processed as in Fig. 2. A large filamentous mitochondrion (m) is seen to be fractured in 3 places (arrows). Other cytoplasmic organelles include: vacuoles (v) and endoplasmic reticulum (er). Part of the nucleus with its nuclear envelope (ne), nucleolus (mi) and heterochromatin (he) are also visible. The euchromatin is highly dispersed in this nucleus, x Fig. 4. A scanning electron micrograph of portions of two uncoated cells in mitosis from a corn root tip osmium ligated and fractured as in Figs. 2 and 3. The upper cell is in metaphase and shows the condensed chromosomes (c) surrounded with less-dense material. The lower cell is in mid-prophase and shows condensed chromosomes (c), nucleolus (mi) and nuclear envelope (ne). x 8500.

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9 Cell organdies viewed with SEM 55 extending parallel with the myofibrils and spanning part way into the A bands, the myosin filaments also extending parallel with the myofibrils and forming the A bands, the H bands bisecting the A bands and the M lines in turn bisecting the H bands. The membranes of the sarcoplasmic reticulum are visible in both micrographs. While the triads, clearly seen in Fig. 7, are not as obvious in Fig. 8, others of our scanning micrographs show this structure more clearly. DISCUSSION It is worthwhile to consider the possibility that osmium depositions during the ligating step do not take place exclusively at the initial sites of osmium bonding. In all of our work we fix and ligate relatively large (2 mm 3 ) pieces of tissue. The transmission micrographs of Figs. 5 and 7 are from thin sections taken deep within the tissue block. It is clear that during ligation most of the added osmium is deposited at the site where osmium was first bound to cellular components during fixation. For the most part these micrographs are typical of transmission micrographs of thin sections stained by conventional means; however, our transmission micrographs of tadpole tails consistently show fine, randomly distributed grains within the tissue. These are especially clear at high magnification as in Fig. 7. The composition and cause of their appearance is not known. It is possible that they are composed of osmium and that they arise because of inability to remove completely either the osmic acid of the fixative, or the TCH during the ligating step; however, their presence in the animal but not the plant material may reflect differences in the ph or buffer systems used (cacodylate for the animal and phosphate for the plant material). Quite obviously their presence in fractured tissue would tend to degrade the image at the cryofractured surface. It is disappointing that ribosomes and microtubules of the spindle are not visualized by this method. It is reasonable to expect structures of this size (24-27 nm) to be resolved if they are preserved by the procedure with sufficient metal attached. There is evidence that ligation may be the limiting step, in that examination of the ligated material in thin section by TEM revealed that the spindle microtubules of the corn roots are difficult to visualize and they and ribosomes in both the corn and tadpole are poorly defined in comparison to similar material lacking the TCH treatment. Fig. 5. A transmission electron micrograph of portions of cells from the epidermis of a tadpole tail fixed and processed in bulk by the osmium ligation method. The thin section received no additional metallic treatment. Structures visible include: nucleus (n), nuclear envelope (ne), plasma membrane (pm), endoplasmic reticulum (er), mitochondria (m), pigment granules (pg), keratin filaments (kf), desmosomes (d), basal lamina (bl), collagen (co) and intercellular canaliculi (ic). x Fig. 6. A scanning electron micrograph of portions of uncoated cells from the epidermis of a tadpole tail osmium ligated and processed similarly to the plant roots of Figs Labelling as in Fig. 5. x Fig. 6A. An enlarged view of the nucleus of Fig. 6 showing the z unit membranes of the nuclear envelope and nuclear pores (arrows), x

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11 Cell organelles viewed with SEM 57 In regard to the best resolutions attained in our scanning micrographs taken at 20 or 30 kev the thinnest regions of the unit membranes that make up the nuclear envelope of the basal cell nucleus shown in Fig. 6 A were measured at approximately 17 nm. Also, in Fig. 8 the smallest filaments in the I bands of this scanning micrograph were measured at approximately 15 nm. In both of these cases the values approach 1 o nm which is the present limit of our SEM and possibly is a practical limit for this method. Although the material of the I-band regions is known to be composed of actin filaments of approximately 5 nm diameter, it is assumed that the structures visualized in Fig. 8 represent aggregates of the smaller actin filaments rather than individuals. It should be pointed out that osmium ligation in combination with resin cracking does not preclude getting information about the surface. Though not illustrated here, we found that SEM images of the more usual sort, restricted to surface irregularities such as seen in Fig. 1, may be obtained from our uncoated, ligated samples by either tilting the mounted specimen to steep angles, by using accelerating voltages of 5 kev or less, or by coating with metal after viewing of the uncoated sample is completed. We chose the method derived from Tanaka & lino (1972) to fracture our tissues, but there is good reason to believe that equivalent results could be had by using the method of Humphreys et al. (1974) in which ethanol-infiltrated tissue is fractured after cooling in liquid nitrogen. Our results show that low-temperature fracturing of osmium-ligated tissue is a useful preparative method for biological material to be examined uncoated by SEM. Plant and animal material so prepared can be viewed at low magnification to study the relationships of tissues and cells or to locate cells of particular interest. Selected areas then can be viewed at higher magnification to study details of cell contents at a resolution approaching that of thin-sectioned material viewed by TEM. In addition some 3-dimensional information from below the fractured surface is made available and this can be especially useful as an aid in interpreting complex structure. This research was carried out at Brookhaven National Laboratory under the auspices of the U.S. Energy Research and Development Administration. We are grateful to Mr R. N. Ruffing and Mr Walter J. Geisbusch for technical assistance rendered. Fig. 7. A transmission electron micrograph of relaxed striated muscle from a tadpole tail fixed and processed in bulk by the osmium ligation method. The thin section received no additional metallic treatment. Structures visible include: Z lines (Z) marking the limits of the sarcomere of each myofibril, I bands (/) with actin filaments extending parallel with the myofibrils and spanning into the A bands (^4), the myosin filaments also extending parallel with the myofibrils and forming the A bands, the H bands (H) bisecting the A bands and the M lines (M) in turn bisecting the H bands. The sarcoplasmic reticulum (sr) and triads or tubular system (ts) are also visible, x Fig. 8. A scanning electron micrograph of uncoated relaxed striated muscle from a tadpole tail osmium ligated twice but otherwise processed similarly to the tissues illustrated in Figs. 2-4 and 6. x

12 58 P. S. Woods and M. C. Ledbetter REFERENCES ANDERSON, T. F. (195I). Techniques for the preservation of three dimensional structures in specimens for the electron microscope. Trans. N.Y. Acad. Set. 13, GUTTMAN, H. N. & STYSKAL, R. C. (1971). Preparation of suspended cells for SEM examination of internal cellular structures. In Scanning Electron Microscopy/i97i, 4th A. Proc. IIT Res. Inst., Chicago, 111., pp HUMPHREYS, W. J., SPURLOCK, B. O. & JOHNSON, J. S. (1974). Critical point drying of ethanolinfiltrated, cryofractured biological specimens for scanning electron microscopy. In Scanning Electron Microscopy/1974, 7th A. Proc. IIT Res. Inst., Chicago, 111., pp HUMPHREYS, W. J. & WODZICKI, T. J. (1972). Methods for viewing by scanning electron microscopy the interior organization of protoplasts of plant cells. In 30th A. Proc. Electron Microsc. Soc. Am. (ed. C. J. Arceneaux), pp , Los Angeles, California. KELLEY, R. O., DEKKER, R. A. & BLUEMINK, J. G. (1973). Ligand-mediated osmium binding: its application in coating biological specimens for scanning electron microscopy. J. Ultrastruct. Res. 45, LIM, D. J. (1971). Scanning electron microscopic observation on non-mechanically cryofractured biological tissue. In Scanning Electron Microscopy/1971, 4th A. Proc. IIT Res. Inst., Chicago, 111., pp LUFT, H. J. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, PANESSA, B. J. & GENNARO, J. F. (1972). A method for direct observation of botanical tissue and intracellular contents by SEM. In 30th A. Proc. Electron Microsc. Soc. Am. (ed. C. J. Areceneaux), pp Los Angeles, California. PANESSA, B. J. & GENNARO, J. F. (1973). Use of potassium iodide/lead acetate for examining uncoated specimens. In Scanning Electron Microscopy/1973, 6th A. Proc. IIT Res. Inst., Chicago, 111., PORTER, K. R., KELLEY, D. & ANDREWS, P. M. (1972). The preparation of cultured cells and soft tissues for scanning electron microscopy. In 5th A. Stereoscan Colloquium, Kent Cambridge Scientific Co., Morton Grove, 111., pp SELIGMAN, A. M., WASSERKRUG, H. L. & HANKER, J. S. (1966). A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxidefixed tissue with osmiophilic thiocarbohydrazide (TCH). J. Cell Biol. 30, TANAKA, K. & IINO, A. (1972). Frozen resin cracking method for scanning electron microscopy and its application to cytology. In 30^ A. Proc. Electron Microsc. Soc. Am. (ed. C. J. Arceneaux), pp Los Angeles, California. WOODS, P. S. & LEDBETTER, M. C. (1974). A method of direct visualization of plant cell organelles for scanning electron microscopy. In 32nd A. Proc. Electron Microsc. Soc. Am. (ed. C. J. Arceneaux), pp St Louis, Missouri. (Received 13 September 1975)