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The fixation of pulmonary surfactant for electron microscopy
© 1969 by Academic Press, It:c.
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J. ULTRASTRUCTURE RESEARCH
27, 88-104 (1969)
The Fixation of Pulmonary Surfactant for Electron Microscopy ~,2 I. The Alveolar Surface Lining Layer GERALD B. DERMER3
Department of Pediatrics, University of California, Los Angeles, California 90024 Received November 15, 1968 The epithelial surface of the lung is thought to be lined by a layer of surfaceactive (surfactant) material. To date, however, there has not been a convincing morphologic demonstration of such an alveolar surface lining layer. Both the major and surface-active component of this material is a saturated phospholipid--dipalmitoyl lecithin. Since standard electron microscopic fixation techniques cannot be used to fix fully saturated lipids, a tissue fixation scheme has been developed which is effective regardless of whether the phospholipids are saturated or unsaturated. This technique (tricomplex flocculation by means of suitable ions) has permitted the electron microscopic visualization of the phospholipid component of surfactant within lung tissue. After tricomplex fixation, a layer of electron dense, polymorphic material is seen lining the epithelial surfaces of alveoli. The epithelial surface of the lung is thought to be lined by a layer of surface-active (surfactant) material. To date, however, there has not been a morphologic demonstration of such an alveolar lining (15) although biochemical studies indicate strongly that such a layer is present (7, 8, 14, 24, 29, 30, 37, 40, 43). This surface-active material has been found to be a phospholipid (30, 40) which contains 70 % saturated fatty acids (29). More recent work has shown the surface-active material to be predominantly dipalmitoyl lecithin (7, 23, 24, 37, 43). These biochemical data probably indicate the reason why electron microscopic studies of osmium-fixed, plastic-embedded lung tissue have either not revealed the presence of a layer of material lining the epithelial surface of the lung (3, 5, 6, 10, 13, 1 A preliminary report of this investigation was presented at the Eighth Meeting of the American Society for Cell Biology in Boston, November, 1968. 2 Supported in part by funds from the United States Public Health Service, the Tuberculosis and Health Association of California, the Los Angeles County Heart Association, and the National Cystic Fibrosis Research Foundation. Present address: Electron Microscopy Laboratory, Department of Pathology, Good Samaritan Hospital, Los Angeles, California 90017.
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25, 32, 38, 45, 50) or have presented unconvincing morphological data supporting the concept of such a layer (22, 27, 28, 31, 36, 47). Osmium tetroxide used as a fixative reacts across the double bonds of unsaturated fatty acids producing osmic acid esters (17). Saturated neutral lipids and phospholipids do not react with OsO~ (1, 12, 26, 42, 44). Moreover, aldehydes cannot be used to fix either saturated or unsaturated lipids. Therefore, since the major component of the alveolar surface lining layer is a saturated phospholipid, it will not be fixed by OsQ and glutaraldehyde, but will be extracted from the tissue during the tissue dehydration in an organic solvent. In addition, unfixed lipids are dissolved by the usual embedding media employed in electron microscopy (11). It seems reasonable to conclude therefore, that the absence of an alveolar surface lining layer in tissue prepared routinely for electron microscopy could be due to its removal from the lung during preparation of the tissue for electron microscopy. With these considerations in mind, a tissue fixative method for phospholipids has been developed which is effective regardless of whether the phospholipids are saturated or unsaturated. This method (tricomplex fiocculation by means of suitable ions) has been used by Elbers et al. (21) to fix emulsions of saturated and unsaturated phospholipids for electron microscopy and is based on the earlier work of Bungenberg De Jong and Saubert (9). The flocculation represents the result of the interaction between three components, an amphoion (the phospholipid), a cation, and an anion. Various salts contain suitable anions and cations. Ion pairs of the cation and anion form links between the phospholipid amphoions, and electrostatic interactions are responsible for the cohesion between phospholipid molecules. Tricomplex flocculation enhances the coherence of the lipid molecules to such an extent that these molecules are not extracted during the preparative treatment necessary for electron microscopy (21). If suitable heavy metal salts are employed, the presence of phospholipids in the tissue should be detected with the electron microscope by the deposition of heavy metal ions at the polar groups of the phospholipid molecules. The observations presented here on lung tissue from breathing, mature guinea pigs prefixed in glutaraldehyde then treated by means of tricomplex flocculation and then postfixed in osmium tetroxide show that the phospholipid component of pulmonary surfactant is easily visualized in sections of acetone dehydrated, plastic embedded tissue. In this report, observations of a layer of electron dense material seen lining the epithelial surfaces of the alveoli (the so-called alveolar surface lining layer) will be discussed. MATERIALS AND METHODS Mature breathing guinea pigs were sacrificed by the injection of 1.0 ml of a 2 % xylocaine solution into the cisternal cavity. Small pieces of lung were then quickly removed and pre-
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fixed for 1 hour in a 2 % glutaraldehyde solution in cacodylate buffer (pH 7.2). The tissue was then rinsed briefly in buffer and then treated by means of tricomplex flocculation for approximately 30 min by placing the tissue in a solution made 0.05 N in Pb(NO3) 2 and K3Fe (CN)6. The tissue was then rinsed again and postfixed in a 1% OsO 4 solution in cacodylate buffer for 1 hour. With other pieces, fixation was carried out by means of glutaraldehyde and tricomplex flocculation only. All tissue was then dehydrated quickly in graded acetones and embedded in Vestopal W. For controls, fixation by means of tricomplex flocculation was eliminated and pieces of lung from the same animal were fixed in only glutaraldehyde and OsO 4. Sections were cut with glass knives on a LKB Ultrotome. Sections from tissue used as controls were usually double stained with uranyl acetate (48) and lead citrate (41), while the sections from tissue treated by means of tricomplex flocculation were not stained to eliminate the heavy metal precipitates that can occur with section staining. The sections were examined in a Hitachi HU-11 A electron microscope at an electron optical magnification of 9000-34,500 times. The micrographs were enlarged photographically as needed. RESULTS Figures 1 and 2 are representative of lung tissue that has been fixed with glutaraldehyde and OsO4, but not by means of tricomplex flocculation. In the first figure, the general organization of the air-blood barrier is observed at low magnification. Adjacent to the alveolar space is a thin cytoplasmic process of an alveolar epithelial cell. Below this, the alveolar epithelium and capillary endothelium are separated by a basement m e m b r a n e of varying width containing granular material. Within the thin cytoplasmic process of the capillary endothelial cell, a few vesicles can be seen. Some granular material is present within the capillary lumen. Fig. 2 is a picture, at higher magnification, of the air-blood barrier. The cell membranes of the alveolar epithelial and capillary endothelial cells are clearly triplelayered structures. Within the cytoplasm of both cell types, m a n y smooth-surfaced vesicles are observed having bounding membranes generally similar in dimensions and density of their layers to the cell membranes of the epithelial and endothelial cells. F o r the most part, the vesicles are empty, but some do contain a finely granular semiopaque material. Some of the vesicles appear to be in different stages of fusion and fission with the cell membranes bounding the alveolar and capillary lumens and also with the cell membranes bounding the basement membrane. A small portion of
FIG. 1. The air-blood barrier from lung tissue fixed in glutaraldehyde and osmium, but not by tricomplex flocculation. There is no material lining the alveolar epithelium. AS, alveolar space; CL, capillary lumen; En, capillary endothelium; Ep, alveolar epithelium; BM, basement membrane. Section stained with uranyl acetate and lead citrate, x 22,500. FrG. 2. A higher magnification view of tissue treated as in Fig. 1. No electron dense material is seen lining the alveolar epithelium. A small part of a red blood cell (RBC) can be seen in the lower portion of the micrograph. Section stained with uranyl acetate and lead citrate. × 86,000.
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a red blood cell is seen in the lower portion of the micrograph. In both Figs. 1 and 2, small particles approximately 50 A in diameter are seen deposited on some cytoplasmic areas and also deposited on regions of the sections outside the boundaries of the tissue. These particles can be identified as lead precipitates which do occur during section staining (I8). Of primary importance is the observation that no material corresponding to surfactant phospholipid can be seen lining the epithelial surfaces of alveoli in tissue that has been fixed only in glutaraldehyde and osmium. In lung tissue that has been treated by means of tricomplex flocculation in addition to OsO~ and glutaraldehyde, reaction product (in the form of electron dense deposits and lamellar structures) is found localized primarily to several distinct areas within the air-blood barrier. Most of the electron dense deposits are found lining the epithelial surfaces of the alveoli attached to the cell membranes of the epithelial cells. A considerable amount of material is also found within the basement membrane that separates the epithelial and endothelial cellular layers. Moreover, smooth-surfaced vesicles within type I alveolar epithelial cells and to a lesser extent within capillary endothelial cells are found to contain similar electron dense material. Reaction product is also seen within inclusion bodies of the type II alveolar cells. Observations presented here will confine themselves to the electron dense material seen lining the epithelial surfaces of the alveoli. In Fig. 3, electron dense globules approximately 0.15-0.3 ,u in diameter are seen forming in some places an almost continuous epithelial lining layer. In other places, the alveolar surface is devoid of any dense material. A few similarly appearing particles are observed free in the lumen of the alveolus. Some of the particles contain highly dense rounded centers with less dense periphery. In another low magnification picture, Fig. 4, the electron dense material forms a continuous layer up to 0.35/~ thick lining the surface of an epithelial cell. Figures 5 and 6 are higher magnification views of portions of tissue shown in Fig. 3. Both pictures show the material lining the surface of the alveolar epithelial cells, which appears primarily in the form of globules with rounded highly dense centers. In places, the cell membranes of the epithelial cells can be identified under the layer of electron opaque material. This is particularly evident in Fig. 7, where a continuous layer of material is seen attached to the outer layer of the cell membrane of an alveolar
FIGS. 3 11. Lung tissue prefixed in glutaraldehyde, then treated by means of tricomplex flocculation and then postfixed in OsO~. Fie. 3. Air-blood barrier. Electron opaque globules, often with intensely dense centers, form in some places an almost continuous epithelial lining layer. Other reaction product is seen as smaller globules primarily within the basement membrane. AS, alveolar space; CL, capillary lumen. Not s~ction stained, x 34,500.
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Fit. 4. A portion of the epithelial side of the air-blood barrier. Electron dense material forms a continuous lining layer on the surface of an epithelial cell. A portion of the cell's nucleus (N) can be seen in the lower part of the micrograph. AS, alveolar space. Not section stained, x 34,500.
epithelial cell. C o n t r a s t within the cytoplasmic areas of these sections is less t h a n that of the controls (Figs. 1 a n d 2) because tissue treated by m e a n s of tricomplex flocc u l a t i o n was n o t section stained with u r a n i u m a n d lead salts. Besides the alveolar surface lining layer, dense deposits of different sizes can be seen over several cytoplasmic areas a n d the b a s e m e n t m e m b r a n e a n d are the reaction p r o d u c t of the triFIGS. 5 and 6. Higher magnification views of portions of tissue shown in Fig. 3. The material lining the surface of the alveolar epithelium can be seen clearly. It appears primarily in the form of globules with rounded intensely staining centers. Also included in both figures are portions of an alveolar epithelial cell (Ep), the basement membranes (Bin) and a capillary endothelial cell (En). Not section stained. AS, alveolar space; CL, capillary lumen. × 103,500.
AS
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Flo. 7. A portion of the epithelial side of the air-blood barrier. The cell membrane of the epithelial cell can be clearly seen under the continuous layer of electron opaque material. Not section stained. x 103,500.
complex fixation of phospholipid localized to other regions of the air-blood barrier besides the surfaces of the epithelial cells. In Figs. 8 and 9, an almost continuous layer of electron dense material can be seen lining the surface of the alveolar epithelial cells, and in Fig. 9, reaction product is present within an invagination of the cell membrane. In these micrographs, as in Figs. 4 and 7, there is practically no electron dense deposit seen over the cellular portions of the air-blood barrier. Sometimes, instead of globular material lining the surfaces of alveolar epithelial cells, lamellar structures consisting of alternating light and dark lines are seen (Fig. 10). The repeat distance of these lamellar structures is approximately 50 A. Periodic lamellar structures also appear to be continuous with the globular material that lines the alveolar surface (Fig. 11). At this magnification, the globular material appears to be composed of rounded electron dense granules approximately 50-100 A in diameter. Fig. 12 is from tissue fixed by glutaraldehyde and tricomplex flocculation, but not by postfixation in osmium. The tissue lacks contrast, but globular material is seen lining the alveolar surface and nearby within the alveolar lumen. As opposed to the situation when tricomplex fixation is followed by osmium tetroxide, the centers of Fins. 8 and 9. An almost continuous layer of electron dense material can be seen lining the surface of the alveolar epithelial cells. Almost no electron dense material is seen over the cellular portions of the air-blood barrier. In Fig. 8, the intensely staining centers of the globules are evident, and in Fig. 9, reaction product is present within an invagination of the cell membrane (arrow). In both figures, the epithelial (Ep), endothelial (En), and basement membrane (BM) portions of the airblood barrier can be clearly seen. In the left-hand part of Fig. 9, there is a small portion of a red blood cell (RBC) within a capillary lumen. AS, alveolar space. Not section stained. × 91,000.
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some of the globules appear less dense than their periphery. Except for the centers of the globules, osmium does not seem to add much contrast to the alveolar surface lining layer, although its effect on the tissue is quite marked. Furthermore, the morphology of the surface material appears quite similar with or without postfixation in osmium (compare Figs. 6 and 12). In Fig. 12, there are several instances where the exteriors of the globules are bounded by dark lines.
DISCUSSION In agreement with Clements (16), electron micrographs of alveolar tissue prepared by standard methods have failed to show any layer external to the alveolar epithelial cells, but the demonstration of such a layer by a new fixation method has been possible. A method for staining chromatograms of phospholipids, tricomplex flocculation by means of suitable ions, has been applied to the fixation of lung tissue for electron microscopy. The mode of action of this fixation, in enhancing the coherence of phospholipid molecules regardless of their degree of saturation, is well known and has allowed the electron microscopic visualization of the saturated phospholipid, dipalmitoyl lecithin, the major component of the alveolar surface lining layer. As opposed to lung tissue that is fixed only in glutaraldehyde and osmium, when additionally tricomplex fixation is employed, electron dense deposits are present as a layer on the surface of alveolar epithelial cells. This material corresponds to the complexes formed between the heavy metal salts used in tricomplex flocculation and phospholipid. The thickness of this layer is variable from about 100 A to more than 0.5/z. The morphology of these deposits strongly supports the concept that this material is phospholipid and suggests that the molecules can be arranged into several structural phases. The tricomplex reaction is specific for phospholipid and unlike osmium does not alter the structure of the lipid molecules (21). Therefore, any electron dense deposits seen within the tissue after tricomplex fixation, if not heavy metal salt precipitates, should be phospholipid visualized by the presence of heavy metal ions at the polar groups of the molecules. It appears that the electron dense material seen in the lung is not simply precipitates of the heavy metal salts used in the fixation scheme, for these deposits are not randomly distributed but localized to a few well defined regions FIG. 10. Lamellar structures consisting of alternating light and dark lines are seen lining the surface of an alveolar epithelial cell (Ep). AS, alveolar space. Not section stained, x 240,000. FIG. 1I. Lamellar structures appear to be continuous with the globular material that lines the alveolar epithelium (Ep). At high magnification, the globular material appears to be composed of small, round granules approximately 50-100 ~ in diameter. AS, alveolar space. Not section stained. x 240,000.
10
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of the air-blood barrier. Most of this material forms a layer lining the surface of the alveolar epithelium, a site where surfactant phospholipid is expected. Reaction product is also located within several defined cytoplasmic regions of the air-blood barrier and might depict stages in the transport of surfactant phospholipid away from the alveolar surface (19). Tricomplex fixation of aqueous dispersions of pure dipalmitoyl lecithin (19) and other phospholipids (21) and tricomplex fixation of surfactant isolated from mammalian lungs (19) produces similar structures to those seen lining the alveolar surface. Moreover, after osmium fixation of aqueous dispersions of unsaturated phospholipid (46) and after negative staining of similar preparations (4, 33), structures are observed which are consistent with the morphology of the alveolar surface lining layer as seen after tricomplex fixation. These data strongly indicate that the material lining the alveolar surface after tricomplex fixation is phospholipid. In phospholipid-water systems, many liquid-crystalline structures exist dependent on lipid concentration and temperature (34). It seems interesting therefore, that the material lining the alveolar surface appears to exist in at least two different physical states. Most characteristically, the surfactant appears as globules often with intensely staining rounded centers after postfixation in osmium. At higher magnification, the globules seem to be made up of small rounded particles 50-100/~ in diameter. In this case, the phospholipid molecules appear to be associated as micelles for a micellar solution of phospholipid does occur in the phase diagram of a lecithin-water system at room temperature above the critical micellar concentration and below the concentration where liquid-crystalline and gel structures are found (35). The highly electron dense centers of the globules which appear after tricomplex, osmium fixation seem to be regions that contain high concentrations of osmium for after tricomplex fixation alone, the centers of the globules are less dense than more peripheral areas. These regions might contain a high proportion of the saturated hydrocarbon chains of the surfactant phospholipid molecules for osmium tetroxide, although it does not react with saturated lipids, it does dissolve within them (2). This is consistent with the observation that after tricomplex fixation alone, the more peripheral regions of the globules are the most electron dense areas and should correspond to regions containing the highest proportion of phospholipid polar groups. If osmium is dissolved within the centers of the globules, it would be reduced during the dehydrations and could be visualized in the sections as deposits of metallic osmium. Alternatively, the centers of the globules could contain a component which chemically reacts with osmium and this component could be protein, for pulmonary surfactant does contain some protein (30, 40) which reacts with osmium (20). According to Luzzati and Husson (34), in the region of concentration between
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FIG. 12. A portion of the epithelial side of the air-blood barrier from lung tissue fixed by glutaraldehyde and tricomplex flocculation, but not by postfixation in osmium. The tissue lacks contrast, but globular material is present at the alveolar surface and nearby within the alveolar lumen. The centers of some of the globules appear less dense than their periphery (arrows). Dark lines bound the exteriors of some of the material (arrows). AS, alveolar space; Ep, alveolar epithelium. Not section stained. × 103,500. the micellar and gel phases, aqueous dispersions of brain phospholipid exhibit lamellar and hexagonal structures, although lecithin dispersions alone display only the lamellar phase (35). By X-ray analysis, the repeat distance of these lamellar structures was found to be 50-60 •, which agrees with the repeat distance of lamellar structures observed in alveolar surface lining material after tricomplex fixation. Consistent with the behavior of lipid-water systems, and in agreement with X-ray analysis of these systems, the alveolar surface lining material displays p o l y m o r p h i s m by exhibiting what appears to be a micellar and lamellar phase. These structures are
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often in close proximity to each other, conceivably due to the different lipid concentrations at these sites. The thickness of the lining layer varies considerably from one region of the alveolar surface to another. In some regions there is no material, and in other areas the layer can vary from about 100 ~ to more than 0.5/~. It seems quite possible that some of this material could have been physically displaced from the alveolar surface after fixation due to the action of the various preparative solutions. It is also not clear whether the fixative penetrated the tissue sufficiently far to react with all the phospholipid material, although tissue taken for analysis was usually near surfaces where the fixative was applied. Evidence presented here suggests that at least in some areas the surfactant layer can be quite thick. However, it is not known whether surfactant is uniformly distributed over the alveolar surface. It is also not known whether tricomplex flocculation has any effect on the native arrangement of the phospholipid molecules for the salt ions used in the fixation scheme, and water might alter the phases in which the lipid is found. This information could be obtained by following the course of the tricomplex reaction with X-ray diffraction. However, except for increasing the electron density of the centers of the globules, osmium postfixation of tricomplex-treated tissue does not seem to alter the morphology of the surface lining layer, for preparations with or without osmium treatment appear quite similar. This would be expected, since the major component of surfactant does not react with osmium. To be surface active, the phospholipid molecules should be in an ordered configuration (16) most suggestive of the lamellar phase. It is encouraging therefore that after tricomplex fixation, lamellar forms are seen within the surface lining layer material. Furthermore, lamellar forms are usually associated with the surface of the surfactant material. The single dark lines seen at the exterior of surfactant deposits could be individual leaflets of dipalmitoyl lecithin, the dark lines corresponding to the heavy metal salts situated at the polar groups of the molecules. The periodic lamellar structures could represent layers of bimolecular leaflets of surfactant phospholipid. These structures would probably be the surface active components of surfactant. Most of the material found below the surface appears primarily in a micellar configuration and could be the so-called "lining complex" of Pattle (39) from which the exterior layer of surface-active molecules could come upon movement to the surface and after a phase change to the lamellar form. Other investigators (22, 28, 31, 47) have described periodic structures within the alveolar lumen and have associated them with surfactant; in one report, what was considered to be an osmiophilic alveolar lining layer was described (27). In these reports, either osmium or osmium in combination with glutaraldehyde was used as the fixative and, as discussed earlier, could not be expected to preserve the saturated
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phospholipid dipalmitoyl lecithin, both the major and the surface active component of the alveolar lining layer material. Furthermore, the repeat distances of the various lamellar forms described are not consistent with figures from aqueous dispersions of phospholipid analyzed in several different ways (4, 33, 35, 46). In the case where an osmiophilic alveolar lining layer was described, it could not be distinguished from the cell membrane of the alveolar epithelial cell. The periodic structures seen in the alveolar lumen after osmium or osmium-glutaraldehyde fixation are probably protein. Any unsaturated lipid components of pulmonary surfactant would also react with osmium. In a very recent attempt to preserve lung surfactant, glutaraldehyde was administered in vivo by perfusion through the lungs' vascular system followed by osmium postfixation (49). However, regardless of how it is applied, osmium and glutaraldehyde will not react with the major component of pulmonary surfactant, dipalmitoyl lecithin. It must be stated, however, that lung surfactant has probably been visualized by Chase (13). When freeze-drying was used instead of any chemical fixatives, a noncellular layer was seen on the surface of alveoli. The author is grateful to Dr. Forrest H. Adams for his support of this project and for the use of the facilities of his laboratory. Thanks are also due to Professor Fritiof S. Sj6strand and Dr. Harry B. Neustein for their critical evaluation of the manuscript.
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27, 88-104 (1969)
The Fixation of Pulmonary Surfactant for Electron Microscopy ~,2 I. The Alveolar Surface Lining Layer GERALD B. DERMER3
Department of Pediatrics, University of California, Los Angeles, California 90024 Received November 15, 1968 The epithelial surface of the lung is thought to be lined by a layer of surfaceactive (surfactant) material. To date, however, there has not been a convincing morphologic demonstration of such an alveolar surface lining layer. Both the major and surface-active component of this material is a saturated phospholipid--dipalmitoyl lecithin. Since standard electron microscopic fixation techniques cannot be used to fix fully saturated lipids, a tissue fixation scheme has been developed which is effective regardless of whether the phospholipids are saturated or unsaturated. This technique (tricomplex flocculation by means of suitable ions) has permitted the electron microscopic visualization of the phospholipid component of surfactant within lung tissue. After tricomplex fixation, a layer of electron dense, polymorphic material is seen lining the epithelial surfaces of alveoli. The epithelial surface of the lung is thought to be lined by a layer of surface-active (surfactant) material. To date, however, there has not been a morphologic demonstration of such an alveolar lining (15) although biochemical studies indicate strongly that such a layer is present (7, 8, 14, 24, 29, 30, 37, 40, 43). This surface-active material has been found to be a phospholipid (30, 40) which contains 70 % saturated fatty acids (29). More recent work has shown the surface-active material to be predominantly dipalmitoyl lecithin (7, 23, 24, 37, 43). These biochemical data probably indicate the reason why electron microscopic studies of osmium-fixed, plastic-embedded lung tissue have either not revealed the presence of a layer of material lining the epithelial surface of the lung (3, 5, 6, 10, 13, 1 A preliminary report of this investigation was presented at the Eighth Meeting of the American Society for Cell Biology in Boston, November, 1968. 2 Supported in part by funds from the United States Public Health Service, the Tuberculosis and Health Association of California, the Los Angeles County Heart Association, and the National Cystic Fibrosis Research Foundation. Present address: Electron Microscopy Laboratory, Department of Pathology, Good Samaritan Hospital, Los Angeles, California 90017.
ELECTRON MICROSCOPY OF PULMONARY SURFACTANT
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25, 32, 38, 45, 50) or have presented unconvincing morphological data supporting the concept of such a layer (22, 27, 28, 31, 36, 47). Osmium tetroxide used as a fixative reacts across the double bonds of unsaturated fatty acids producing osmic acid esters (17). Saturated neutral lipids and phospholipids do not react with OsO~ (1, 12, 26, 42, 44). Moreover, aldehydes cannot be used to fix either saturated or unsaturated lipids. Therefore, since the major component of the alveolar surface lining layer is a saturated phospholipid, it will not be fixed by OsQ and glutaraldehyde, but will be extracted from the tissue during the tissue dehydration in an organic solvent. In addition, unfixed lipids are dissolved by the usual embedding media employed in electron microscopy (11). It seems reasonable to conclude therefore, that the absence of an alveolar surface lining layer in tissue prepared routinely for electron microscopy could be due to its removal from the lung during preparation of the tissue for electron microscopy. With these considerations in mind, a tissue fixative method for phospholipids has been developed which is effective regardless of whether the phospholipids are saturated or unsaturated. This method (tricomplex fiocculation by means of suitable ions) has been used by Elbers et al. (21) to fix emulsions of saturated and unsaturated phospholipids for electron microscopy and is based on the earlier work of Bungenberg De Jong and Saubert (9). The flocculation represents the result of the interaction between three components, an amphoion (the phospholipid), a cation, and an anion. Various salts contain suitable anions and cations. Ion pairs of the cation and anion form links between the phospholipid amphoions, and electrostatic interactions are responsible for the cohesion between phospholipid molecules. Tricomplex flocculation enhances the coherence of the lipid molecules to such an extent that these molecules are not extracted during the preparative treatment necessary for electron microscopy (21). If suitable heavy metal salts are employed, the presence of phospholipids in the tissue should be detected with the electron microscope by the deposition of heavy metal ions at the polar groups of the phospholipid molecules. The observations presented here on lung tissue from breathing, mature guinea pigs prefixed in glutaraldehyde then treated by means of tricomplex flocculation and then postfixed in osmium tetroxide show that the phospholipid component of pulmonary surfactant is easily visualized in sections of acetone dehydrated, plastic embedded tissue. In this report, observations of a layer of electron dense material seen lining the epithelial surfaces of the alveoli (the so-called alveolar surface lining layer) will be discussed. MATERIALS AND METHODS Mature breathing guinea pigs were sacrificed by the injection of 1.0 ml of a 2 % xylocaine solution into the cisternal cavity. Small pieces of lung were then quickly removed and pre-
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fixed for 1 hour in a 2 % glutaraldehyde solution in cacodylate buffer (pH 7.2). The tissue was then rinsed briefly in buffer and then treated by means of tricomplex flocculation for approximately 30 min by placing the tissue in a solution made 0.05 N in Pb(NO3) 2 and K3Fe (CN)6. The tissue was then rinsed again and postfixed in a 1% OsO 4 solution in cacodylate buffer for 1 hour. With other pieces, fixation was carried out by means of glutaraldehyde and tricomplex flocculation only. All tissue was then dehydrated quickly in graded acetones and embedded in Vestopal W. For controls, fixation by means of tricomplex flocculation was eliminated and pieces of lung from the same animal were fixed in only glutaraldehyde and OsO 4. Sections were cut with glass knives on a LKB Ultrotome. Sections from tissue used as controls were usually double stained with uranyl acetate (48) and lead citrate (41), while the sections from tissue treated by means of tricomplex flocculation were not stained to eliminate the heavy metal precipitates that can occur with section staining. The sections were examined in a Hitachi HU-11 A electron microscope at an electron optical magnification of 9000-34,500 times. The micrographs were enlarged photographically as needed. RESULTS Figures 1 and 2 are representative of lung tissue that has been fixed with glutaraldehyde and OsO4, but not by means of tricomplex flocculation. In the first figure, the general organization of the air-blood barrier is observed at low magnification. Adjacent to the alveolar space is a thin cytoplasmic process of an alveolar epithelial cell. Below this, the alveolar epithelium and capillary endothelium are separated by a basement m e m b r a n e of varying width containing granular material. Within the thin cytoplasmic process of the capillary endothelial cell, a few vesicles can be seen. Some granular material is present within the capillary lumen. Fig. 2 is a picture, at higher magnification, of the air-blood barrier. The cell membranes of the alveolar epithelial and capillary endothelial cells are clearly triplelayered structures. Within the cytoplasm of both cell types, m a n y smooth-surfaced vesicles are observed having bounding membranes generally similar in dimensions and density of their layers to the cell membranes of the epithelial and endothelial cells. F o r the most part, the vesicles are empty, but some do contain a finely granular semiopaque material. Some of the vesicles appear to be in different stages of fusion and fission with the cell membranes bounding the alveolar and capillary lumens and also with the cell membranes bounding the basement membrane. A small portion of
FIG. 1. The air-blood barrier from lung tissue fixed in glutaraldehyde and osmium, but not by tricomplex flocculation. There is no material lining the alveolar epithelium. AS, alveolar space; CL, capillary lumen; En, capillary endothelium; Ep, alveolar epithelium; BM, basement membrane. Section stained with uranyl acetate and lead citrate, x 22,500. FrG. 2. A higher magnification view of tissue treated as in Fig. 1. No electron dense material is seen lining the alveolar epithelium. A small part of a red blood cell (RBC) can be seen in the lower portion of the micrograph. Section stained with uranyl acetate and lead citrate. × 86,000.
AS
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a red blood cell is seen in the lower portion of the micrograph. In both Figs. 1 and 2, small particles approximately 50 A in diameter are seen deposited on some cytoplasmic areas and also deposited on regions of the sections outside the boundaries of the tissue. These particles can be identified as lead precipitates which do occur during section staining (I8). Of primary importance is the observation that no material corresponding to surfactant phospholipid can be seen lining the epithelial surfaces of alveoli in tissue that has been fixed only in glutaraldehyde and osmium. In lung tissue that has been treated by means of tricomplex flocculation in addition to OsO~ and glutaraldehyde, reaction product (in the form of electron dense deposits and lamellar structures) is found localized primarily to several distinct areas within the air-blood barrier. Most of the electron dense deposits are found lining the epithelial surfaces of the alveoli attached to the cell membranes of the epithelial cells. A considerable amount of material is also found within the basement membrane that separates the epithelial and endothelial cellular layers. Moreover, smooth-surfaced vesicles within type I alveolar epithelial cells and to a lesser extent within capillary endothelial cells are found to contain similar electron dense material. Reaction product is also seen within inclusion bodies of the type II alveolar cells. Observations presented here will confine themselves to the electron dense material seen lining the epithelial surfaces of the alveoli. In Fig. 3, electron dense globules approximately 0.15-0.3 ,u in diameter are seen forming in some places an almost continuous epithelial lining layer. In other places, the alveolar surface is devoid of any dense material. A few similarly appearing particles are observed free in the lumen of the alveolus. Some of the particles contain highly dense rounded centers with less dense periphery. In another low magnification picture, Fig. 4, the electron dense material forms a continuous layer up to 0.35/~ thick lining the surface of an epithelial cell. Figures 5 and 6 are higher magnification views of portions of tissue shown in Fig. 3. Both pictures show the material lining the surface of the alveolar epithelial cells, which appears primarily in the form of globules with rounded highly dense centers. In places, the cell membranes of the epithelial cells can be identified under the layer of electron opaque material. This is particularly evident in Fig. 7, where a continuous layer of material is seen attached to the outer layer of the cell membrane of an alveolar
FIGS. 3 11. Lung tissue prefixed in glutaraldehyde, then treated by means of tricomplex flocculation and then postfixed in OsO~. Fie. 3. Air-blood barrier. Electron opaque globules, often with intensely dense centers, form in some places an almost continuous epithelial lining layer. Other reaction product is seen as smaller globules primarily within the basement membrane. AS, alveolar space; CL, capillary lumen. Not s~ction stained, x 34,500.
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Fit. 4. A portion of the epithelial side of the air-blood barrier. Electron dense material forms a continuous lining layer on the surface of an epithelial cell. A portion of the cell's nucleus (N) can be seen in the lower part of the micrograph. AS, alveolar space. Not section stained, x 34,500.
epithelial cell. C o n t r a s t within the cytoplasmic areas of these sections is less t h a n that of the controls (Figs. 1 a n d 2) because tissue treated by m e a n s of tricomplex flocc u l a t i o n was n o t section stained with u r a n i u m a n d lead salts. Besides the alveolar surface lining layer, dense deposits of different sizes can be seen over several cytoplasmic areas a n d the b a s e m e n t m e m b r a n e a n d are the reaction p r o d u c t of the triFIGS. 5 and 6. Higher magnification views of portions of tissue shown in Fig. 3. The material lining the surface of the alveolar epithelium can be seen clearly. It appears primarily in the form of globules with rounded intensely staining centers. Also included in both figures are portions of an alveolar epithelial cell (Ep), the basement membranes (Bin) and a capillary endothelial cell (En). Not section stained. AS, alveolar space; CL, capillary lumen. × 103,500.
AS
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Flo. 7. A portion of the epithelial side of the air-blood barrier. The cell membrane of the epithelial cell can be clearly seen under the continuous layer of electron opaque material. Not section stained. x 103,500.
complex fixation of phospholipid localized to other regions of the air-blood barrier besides the surfaces of the epithelial cells. In Figs. 8 and 9, an almost continuous layer of electron dense material can be seen lining the surface of the alveolar epithelial cells, and in Fig. 9, reaction product is present within an invagination of the cell membrane. In these micrographs, as in Figs. 4 and 7, there is practically no electron dense deposit seen over the cellular portions of the air-blood barrier. Sometimes, instead of globular material lining the surfaces of alveolar epithelial cells, lamellar structures consisting of alternating light and dark lines are seen (Fig. 10). The repeat distance of these lamellar structures is approximately 50 A. Periodic lamellar structures also appear to be continuous with the globular material that lines the alveolar surface (Fig. 11). At this magnification, the globular material appears to be composed of rounded electron dense granules approximately 50-100 A in diameter. Fig. 12 is from tissue fixed by glutaraldehyde and tricomplex flocculation, but not by postfixation in osmium. The tissue lacks contrast, but globular material is seen lining the alveolar surface and nearby within the alveolar lumen. As opposed to the situation when tricomplex fixation is followed by osmium tetroxide, the centers of Fins. 8 and 9. An almost continuous layer of electron dense material can be seen lining the surface of the alveolar epithelial cells. Almost no electron dense material is seen over the cellular portions of the air-blood barrier. In Fig. 8, the intensely staining centers of the globules are evident, and in Fig. 9, reaction product is present within an invagination of the cell membrane (arrow). In both figures, the epithelial (Ep), endothelial (En), and basement membrane (BM) portions of the airblood barrier can be clearly seen. In the left-hand part of Fig. 9, there is a small portion of a red blood cell (RBC) within a capillary lumen. AS, alveolar space. Not section stained. × 91,000.
7 -- 691830
J . Ultra8tructure Research
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some of the globules appear less dense than their periphery. Except for the centers of the globules, osmium does not seem to add much contrast to the alveolar surface lining layer, although its effect on the tissue is quite marked. Furthermore, the morphology of the surface material appears quite similar with or without postfixation in osmium (compare Figs. 6 and 12). In Fig. 12, there are several instances where the exteriors of the globules are bounded by dark lines.
DISCUSSION In agreement with Clements (16), electron micrographs of alveolar tissue prepared by standard methods have failed to show any layer external to the alveolar epithelial cells, but the demonstration of such a layer by a new fixation method has been possible. A method for staining chromatograms of phospholipids, tricomplex flocculation by means of suitable ions, has been applied to the fixation of lung tissue for electron microscopy. The mode of action of this fixation, in enhancing the coherence of phospholipid molecules regardless of their degree of saturation, is well known and has allowed the electron microscopic visualization of the saturated phospholipid, dipalmitoyl lecithin, the major component of the alveolar surface lining layer. As opposed to lung tissue that is fixed only in glutaraldehyde and osmium, when additionally tricomplex fixation is employed, electron dense deposits are present as a layer on the surface of alveolar epithelial cells. This material corresponds to the complexes formed between the heavy metal salts used in tricomplex flocculation and phospholipid. The thickness of this layer is variable from about 100 A to more than 0.5/z. The morphology of these deposits strongly supports the concept that this material is phospholipid and suggests that the molecules can be arranged into several structural phases. The tricomplex reaction is specific for phospholipid and unlike osmium does not alter the structure of the lipid molecules (21). Therefore, any electron dense deposits seen within the tissue after tricomplex fixation, if not heavy metal salt precipitates, should be phospholipid visualized by the presence of heavy metal ions at the polar groups of the molecules. It appears that the electron dense material seen in the lung is not simply precipitates of the heavy metal salts used in the fixation scheme, for these deposits are not randomly distributed but localized to a few well defined regions FIG. 10. Lamellar structures consisting of alternating light and dark lines are seen lining the surface of an alveolar epithelial cell (Ep). AS, alveolar space. Not section stained, x 240,000. FIG. 1I. Lamellar structures appear to be continuous with the globular material that lines the alveolar epithelium (Ep). At high magnification, the globular material appears to be composed of small, round granules approximately 50-100 ~ in diameter. AS, alveolar space. Not section stained. x 240,000.
10
11
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of the air-blood barrier. Most of this material forms a layer lining the surface of the alveolar epithelium, a site where surfactant phospholipid is expected. Reaction product is also located within several defined cytoplasmic regions of the air-blood barrier and might depict stages in the transport of surfactant phospholipid away from the alveolar surface (19). Tricomplex fixation of aqueous dispersions of pure dipalmitoyl lecithin (19) and other phospholipids (21) and tricomplex fixation of surfactant isolated from mammalian lungs (19) produces similar structures to those seen lining the alveolar surface. Moreover, after osmium fixation of aqueous dispersions of unsaturated phospholipid (46) and after negative staining of similar preparations (4, 33), structures are observed which are consistent with the morphology of the alveolar surface lining layer as seen after tricomplex fixation. These data strongly indicate that the material lining the alveolar surface after tricomplex fixation is phospholipid. In phospholipid-water systems, many liquid-crystalline structures exist dependent on lipid concentration and temperature (34). It seems interesting therefore, that the material lining the alveolar surface appears to exist in at least two different physical states. Most characteristically, the surfactant appears as globules often with intensely staining rounded centers after postfixation in osmium. At higher magnification, the globules seem to be made up of small rounded particles 50-100/~ in diameter. In this case, the phospholipid molecules appear to be associated as micelles for a micellar solution of phospholipid does occur in the phase diagram of a lecithin-water system at room temperature above the critical micellar concentration and below the concentration where liquid-crystalline and gel structures are found (35). The highly electron dense centers of the globules which appear after tricomplex, osmium fixation seem to be regions that contain high concentrations of osmium for after tricomplex fixation alone, the centers of the globules are less dense than more peripheral areas. These regions might contain a high proportion of the saturated hydrocarbon chains of the surfactant phospholipid molecules for osmium tetroxide, although it does not react with saturated lipids, it does dissolve within them (2). This is consistent with the observation that after tricomplex fixation alone, the more peripheral regions of the globules are the most electron dense areas and should correspond to regions containing the highest proportion of phospholipid polar groups. If osmium is dissolved within the centers of the globules, it would be reduced during the dehydrations and could be visualized in the sections as deposits of metallic osmium. Alternatively, the centers of the globules could contain a component which chemically reacts with osmium and this component could be protein, for pulmonary surfactant does contain some protein (30, 40) which reacts with osmium (20). According to Luzzati and Husson (34), in the region of concentration between
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FIG. 12. A portion of the epithelial side of the air-blood barrier from lung tissue fixed by glutaraldehyde and tricomplex flocculation, but not by postfixation in osmium. The tissue lacks contrast, but globular material is present at the alveolar surface and nearby within the alveolar lumen. The centers of some of the globules appear less dense than their periphery (arrows). Dark lines bound the exteriors of some of the material (arrows). AS, alveolar space; Ep, alveolar epithelium. Not section stained. × 103,500. the micellar and gel phases, aqueous dispersions of brain phospholipid exhibit lamellar and hexagonal structures, although lecithin dispersions alone display only the lamellar phase (35). By X-ray analysis, the repeat distance of these lamellar structures was found to be 50-60 •, which agrees with the repeat distance of lamellar structures observed in alveolar surface lining material after tricomplex fixation. Consistent with the behavior of lipid-water systems, and in agreement with X-ray analysis of these systems, the alveolar surface lining material displays p o l y m o r p h i s m by exhibiting what appears to be a micellar and lamellar phase. These structures are
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often in close proximity to each other, conceivably due to the different lipid concentrations at these sites. The thickness of the lining layer varies considerably from one region of the alveolar surface to another. In some regions there is no material, and in other areas the layer can vary from about 100 ~ to more than 0.5/~. It seems quite possible that some of this material could have been physically displaced from the alveolar surface after fixation due to the action of the various preparative solutions. It is also not clear whether the fixative penetrated the tissue sufficiently far to react with all the phospholipid material, although tissue taken for analysis was usually near surfaces where the fixative was applied. Evidence presented here suggests that at least in some areas the surfactant layer can be quite thick. However, it is not known whether surfactant is uniformly distributed over the alveolar surface. It is also not known whether tricomplex flocculation has any effect on the native arrangement of the phospholipid molecules for the salt ions used in the fixation scheme, and water might alter the phases in which the lipid is found. This information could be obtained by following the course of the tricomplex reaction with X-ray diffraction. However, except for increasing the electron density of the centers of the globules, osmium postfixation of tricomplex-treated tissue does not seem to alter the morphology of the surface lining layer, for preparations with or without osmium treatment appear quite similar. This would be expected, since the major component of surfactant does not react with osmium. To be surface active, the phospholipid molecules should be in an ordered configuration (16) most suggestive of the lamellar phase. It is encouraging therefore that after tricomplex fixation, lamellar forms are seen within the surface lining layer material. Furthermore, lamellar forms are usually associated with the surface of the surfactant material. The single dark lines seen at the exterior of surfactant deposits could be individual leaflets of dipalmitoyl lecithin, the dark lines corresponding to the heavy metal salts situated at the polar groups of the molecules. The periodic lamellar structures could represent layers of bimolecular leaflets of surfactant phospholipid. These structures would probably be the surface active components of surfactant. Most of the material found below the surface appears primarily in a micellar configuration and could be the so-called "lining complex" of Pattle (39) from which the exterior layer of surface-active molecules could come upon movement to the surface and after a phase change to the lamellar form. Other investigators (22, 28, 31, 47) have described periodic structures within the alveolar lumen and have associated them with surfactant; in one report, what was considered to be an osmiophilic alveolar lining layer was described (27). In these reports, either osmium or osmium in combination with glutaraldehyde was used as the fixative and, as discussed earlier, could not be expected to preserve the saturated
ELECTRON MICROSCOPY OF PULMONARY SURFACTANT
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phospholipid dipalmitoyl lecithin, both the major and the surface active component of the alveolar lining layer material. Furthermore, the repeat distances of the various lamellar forms described are not consistent with figures from aqueous dispersions of phospholipid analyzed in several different ways (4, 33, 35, 46). In the case where an osmiophilic alveolar lining layer was described, it could not be distinguished from the cell membrane of the alveolar epithelial cell. The periodic structures seen in the alveolar lumen after osmium or osmium-glutaraldehyde fixation are probably protein. Any unsaturated lipid components of pulmonary surfactant would also react with osmium. In a very recent attempt to preserve lung surfactant, glutaraldehyde was administered in vivo by perfusion through the lungs' vascular system followed by osmium postfixation (49). However, regardless of how it is applied, osmium and glutaraldehyde will not react with the major component of pulmonary surfactant, dipalmitoyl lecithin. It must be stated, however, that lung surfactant has probably been visualized by Chase (13). When freeze-drying was used instead of any chemical fixatives, a noncellular layer was seen on the surface of alveoli. The author is grateful to Dr. Forrest H. Adams for his support of this project and for the use of the facilities of his laboratory. Thanks are also due to Professor Fritiof S. Sj6strand and Dr. Harry B. Neustein for their critical evaluation of the manuscript.
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