- Email: [email protected]
Electron microscope tomography
TIBS 11 - December 1986
499
Emerging Techniques Electron microscope tomography Ulf Skoglund and Bertil Daneholt Electron microscope tomography allows three-dimensional reconstruction of ultrastructural objects at the molecular level. The method is general and not limited to symmetric, or regularly ordered structures. Alone, or in combination with immunoelectron microscopy and electron spectroscopic imaging, electron microscope tomography is a powerful technique in cell and molecular biology.
Electron microscopy was a breakthrough technique in the analysis of the structure of the cell: cellular organelles already known could be described in a much greater detail, and new organelles and membrane systems were revealed. The subsequent ability to fractionate cells into well defined cellular components has made possible the biochemical study of cell structures, and electron microscopy is often used to verify the nature, quality and purity of a preparation. More recently, application of recombinant D N A technology and related techniques has allowed virtually any eukaryotic gene to be isolated and the corresponding protein prepared in large quantities and characterized. The main problem is usually to establish the function of a given protein, especially as the proteins are frequently components of complex cell structures. Thus, an important task for present-day biochemists is to reconstitute such complex structures from well defined molecular components and to elucidate the function of the constituents. The need for ultrastructural information, both in vivo and /n vitro, to match the development of the biochemical analysis of the cell becomes more and more obvious. Electron microscope tomography is a new technique that can be used to reconstruct ultrastructural objects in three dimensions at the molecular level.
Three-dimensional techniques A three-dimensional image of an electron microscopic specimen-can be obtained by inspecting two different electron micrograph projections of the U. Skogland and B. Daneholt are at the Department of Medical Cell Genetics, Medical Nobel Institute, Karolinska Institute, Box 60400, S-104 Ol Stockholm, Sweden.
object with a stereo viewer I. This procedure is often useful, but does not permit precise and quantitative descriptions of an object in three dimensions. Thus, there has been an urgent need to develop techniques for three-dimensional reconstruction of ultrastructural objects. An electron microscopic image is a two-dimensional projection of a threedimensional structure. By collecting a certain number of different projections of an object, one has sufficient information to accomplish a proper three-
\
i
,;,
dimensional reconstruction (Fig. 1). The mathematical theory for such a reconstruction approach was worked out as long ago as 1917 by J. Radon 2. DeRosier and Klug 3 were the first to apply this theory in electron microscopy when they reconstructed the spiral-shaped segment of phage T4 and made use of its helical symmetry to obtain a reliable reconstruction. The subsequent development of the mathematical and numerical aspects of the theory made it possible to study spherical virus particles 4 as well as molecular entities highly ordered in two dimensions s. These studies of symmetrical, or regularly arranged structures established the three-dimensional reconstruction method as a powerful tool for structural molecular research (for review, see Ref. 6). It is evident that a reconstruction procedure would have much greater applicability if the need to utilize symmetries in the analysis could be overcome. The paramount problem with the analysis of non-symmetric structures is that the different projections are difficult to align precisely with each other (high precision in defining a common origin is crucial for the final resolution of the reconstruction). An early aligning approach was based on the calculation of
I
,.,
" i v
Fig. 1. The principle of three-dimensional image reconstruction from electron micrographs. Five micrographs, shown in a semicircular arrangement, represent a set of projections of a centrally placed specimen which are accurately aligned in relation to each other to allow image reconstruction. The three-dimensional reconstruction takes place as a series of two-dimensional reconstructions: in each single step, one of which is shown in the figure, a circular disc is reconstructed by back-projection (after suitable weighting) of the corresponding lines in the micrographs e7,2~. Note that the lines and the reconstructed disc are perpendicular to the tilt axis. Several consecutive discs ultimately form a cylinder in which the reconstructed object is contained. Reproduced, with permission, from Ref. 10. ~) 1986,ElsevierSciencePublishersB.V.. Amsterdam 0376 5067/86/$02.00
500 cross-correlation functions between successive tilts 7. More recently, colloidal gold particles spread on thin sections have been used as reference points for alignment procedures 8.9. These early studies showed the feasibility of reconstructing an aperiodic object from electron micrographs in a tilt series. Our laboratory has recently worked out a new method for the three-dimensional reconstruction of ultrastructural objects m, symmetric and asymmetric, to a resolution better than 50 A. The origin problem has been solved by the colloidal gold method: the coordinates of small gold particles are determined to calculate image beam rotation, tilt angle, shrinkage, changes of planarity and parallaxes in the whole tilt (projection) series. A coherent computer software package has been put together, tested and is now used routinely. The technique has been applied to ultrathin sections for studies of nucleic acid-protein complexes at molecular dimensions. To demonstrate the power of the method and the nature of the results we will discuss a recent investigation of a specific premessenger ribonucleoprotein (RNP) particle 10.
Experimental material Giant polytene chromosomes in the salivary glands of the midge C h i r o n o m u s tentans were chosen for ultrastructural studies (for review, see Ref. 11). These chromosomes have acquired their exceptional size by juxtaposition of thousands of identical elements, each equivalent to a chromosome in a diploid cell. The chromosomes can be identified easily and specific chromosome regions investigated with electron microscopy. We have concentrated our studies on two selected segments of chromosome IV, the Balbiani rings 1 and 2. These chromosome segments harbour genes coding for large secretory proteins. The Balbiani ring (BR) genes are transcriptionally very active in the salivary glands and have been characterized by electron microscopy both in situ 12-14 and after chromosome IV has been isolated and spread on a surface 15. It has been possible to observe how the large primary transcripts (75S RNA) are synthesized in the BRs andpacked together with proteins into 500 A diameter RNP particles. The abundance and exceptional size of BR particles makes them readily identifiable in the nuclear sap and in the nuclear pores. We have used the tomography method to reconstruct the BR particle in three dimensions during its synthesis, its transport to the nuclear pore and its passage through the pore.
T I B S 11 - D e c e m b e r 1986
Computed tomography technique Ultrathin sections through salivary glands were prepared by conventional procedures, and colloidal gold particles were deposited onto the sections to obtain alignment markers. The specimens were examined in a conventional transmission electron microscope provided with a goniometer to allow tilting. A suitable nuclear area with BR particles was identified and photographed as a series of tilted views (every 10° in the range +60 °) (Fig. 2). Prior to the actual threedimensional reconstruction the electron micrographs were digitized and aligned with an iterative full least-squares procedure using the gold particles as alignment markers (G. Bricogne and U. Skoglund, unpublished). Information was selected from each electron micrograph for the reconstruction (Fig. 3). The three-dimensional reconstruction followed the 'filtered back-projection principle', in which the Fourier transforms of the projections (tilts) are radially weighted 16. As shown in Fig. 1, each three-dimensional reconstruction was composed of a series of two-dimensional reconstructions of circular disks perpendicular to, and repeated along, the tilt axis, forming a cylinder. The completed reconstruction was shown as a number of density maps corresponding to the reconstructed sections through the object. The particle as a whole was presented both as a balsa-wood model (Fig. 3 in Ref. 10) and as a contoured vector image on a display (see Fig. 4).
Three-dimensional structure of the BR RNP particle Four individual BR RNP particles were reconstructed: they displayed strikingly similar gross morphology irrespective of their orientation within the sections. The high correlation coefficients between the various particles (about 0.8) permitted the calculation of an average structure. This average particle is presented in Fig. 4: it has a hole in the centre and a slit at 1-2 o'clock, giving the particle a ring-like appearance. The RNP element is asymmetric and can be described as a thick ribbon divided into four separate domains, designated 1-4 (Fig. 4A). Furthermore, the asymmetry of the particle made it possible to predict the location of the two ends of the R N A molecule. By comparing the features of the growing RNP particle on the gene and the domain organization of the particle, it was concluded that domain 1 is completed first and probably contains the 5' end of the R N A molecule, while domain 4 is the last one to be finished and therefore probably harbours the 3' end. The ring-like structure with the two ends of the R N A molecule close together in the particle can have important functional implications for the splicing, polyadenylation and transport processes (for discussion, see Ref. 10). Resolution of the method The BR RNP particles were reconstructed to a resolution of 80-90 ~ , which is close to the predicted theoretical
!
Fig. 2. Electron micrograph of Balbiani ring R N P particles in the nucleus of a salivary gland cell. The nuclear envelope separates nuclear sap (left) from cytoplasm (right). The B R R N P particles in nuclear sap are encircled, and a large arrow denotes a BR particle being translocated through a nuclear pore. Some of the gold particles serving as alignment markers are indicated by small arrows. The photograph represents a 0 ~ degree tilt in a tilt series comprising 13 photographs covering the --60~ range. The scale bar corresponds to 2000 A. For experimental details, see Ref. 10.
T I B S 11 - D e c e m b e r
501
1986
well as stain densities; separate reconstructions can be compared and differences and similarities evaluated; average structures can be calculated and interpreted; selected segments of a structure can be studied separately and compared with other segments or with other particles to find similar, or complementary, structures; specific elements within a structure can be traced. It is also possible to construct specific models and compare those with the structure obtained. It should be stressed that m o d e m computer graphic techniques offer excellent aids for this type of analysis.
Fig. 3. Thirteen extracts from a tilt series displaying the same B R R N P particle in the centre. Together these extracts represent an aligned set of projected views of the particle and are used as input data for the reconstruction algorithm. The tilt axis runs vertically through the middle o f each extract; the tilt angles have been indicated with labels. A few colloidal gold markers are visible as dark circular objects. Every extract is 3100 fii square. The image was produced on a display and photographed directly on the screen.
limit (85 A.) for a 500 A_diameter object, photographed under optimal conditions in 10° intervals in the range _+90° (Ref. 16). This result suggests that the restriction of tilt angles to _+60° on m o d e m electron microscope goniometers only has a minor effect on the final structure when the object has good contrast. The key to a high resolution in the tomographic procedure is the proper alignment of the electron micrographs comprising the tilt series. For our routine reconstructions today we generally achieve a 6 A. prediction error for any point anywhere in the tilt series. This allows us to get high quality reconstructions of the 500 A particles at a resolution better than 50 A when the tilt series comprises 25 electron micrographs taken at 5 ° intervals between _+60° . It should be noted that smaller objects than the B R particle will be reconstructed at a still higher resolution, because the resolution in a given reconstruction is inversely proportional to the thickness of the stained object 16. Since there is no indication of radiation damage, it seems likely that we can further improve the resolution by increasing the
number of tilted views of the specimen (the resolution is directly proportional to the number of evenly distributed tiltsl6). The ultimate limit for the resolution in a positively stained specimen is set by the stain distribution and has been estimated to be about 20 ,~ (Ref. 17). It is not yet possible to predict how close to this limit the computed tomography can get.
Analytical power In conventional transmission electron microscopy it is often difficult to demarcate a specific structure because of the surrounding background or superimposed structures. These problems are usually circumvented by the threedimensional reconstruction method. Furthermore, in tomography the reconstructed object can be rotated and studied from all directions, not only from those available in the electron microscope. The most essential point is, however, that the tomography approach permits a precise quantitative description and advanced analysis of the object. A given reconstruction can be studied in a number of quantitative ways: volumes and distances can be measured as
Combination of computed tomography with other methods Electron microscope tomography will be most useful when it is combined with methods that provide information on the biochemical nature of the structure. Specific enzyme activities can be detected by local deposition of electron-dense material on the structure (enzyme cytochemistry) TM. A more general and precise approach would be to use specific ligands tagged with electron-dense markers, preferably colloidal gold, in order to locate defined macromolecules at the ultrastructural levellg-2k Antibodies, ideally monoclonals, represent the most obvious and versatile possibility (immunoelectron microscopy), but there are a rapidly increasing number of other ligands available: hormones for their receptors, lectins for specific moieties of glycoproteins and glycolipids, nucleases for their nucleic acid substrates, low density lipoproteins for their membrane receptors, etc. For nucleic acid-containing structures, in situ hybridization could also be an attractive possibility; a recent study of ribosome structure has shown that a short, single-stranded D N A probe labelled with biotin can be hybridized to the corresponding ribosomal R N A sequence and visualized in the electron microscope after addition of avidin 2:. An elemental analysis of the ultrastructural object could be useful in some circumstances. The conventional X-ray microanalysis can provide information on atoms 23 but usually the resolution is too low to be optimal for molecular entities (spot anal)zsis can reach a resolution of about 200 A; Ref. 24). For biological objects the new approach of using scattered electrons to determine the energy loss spectrum (electron energy loss spectroscopy) seems more promisingS7.25; electron spectroscopic imaging is particularly sensitive, with a more than tenfold better resolution than X-ray microanalysis iT. One example of the
502
T I B S 11 - D e c e m b e r 1986
Fig. 4. The average B R R N P structure represented as a three-dimensional vector model. Six views are shown, labelled A - F . Four domains are outlined in A and numbered 1~l; the red line (upper righO demarcates the slit between domain 1 and domain 4. A s a further aid to viewing B-E, the demarcation line and the positions o f domain 1 and 4 are shown. Domain 2 constitutes the major part o f the bottom view o f the structure seen in F. The image was composed and photographed on a
display.
feasibility of the electron spectroscopic imaging method at the molecular level is the elucidation of the path of D N A around the histone octamer of a nucleosome by analysis of the phosphorus distribution in individual nucleosomes 26.
Applicability We have stressed that electron microscope tomography allows the reconstruction of any transparent ultrastructural object in three dimensions. One example has been given from our own research programme, but it is clear that the method has a very wide spectrum of applications in cellular and molecular biology. It should be possible to analyse a variety of subcellular organelles, filamentous structures and large molecular complexes such as ribosomes. The method should be well suited to study dynamic processes such as intracellular transport, receptor-ligand interactions of various kinds and cell-to-ceU contacts.
Computed tomography in itself will provide the distribution of mass in the molecular complexes, and when combined with the use of specific ligands coupled to electron-dense markers it will be a most powerful tool to identify and localize specific macromolecules within the cell. It seems likely that electron microscope tomography will contribute effectively to change the emphasis in cell biology from ultrastructural objects to molecular entities.
Acknowledgements We are grateful to Hans Mehlin and Anna Sj6d6n for stimulating discussions and technical assistance and to Evy Vesterb~ick for typing the manuscript. This research was supported by Knut and Alice Wallenberg Foundation, Swedish Natural Science Research Council and National Swedish Board for Technical Development.
References 1 Turner, J. N. (1981) in Methods in Cell Biology (Vol. 22) (Turner, J. N., ed.), pp. 33-5l, Academic Press 2 Radon, J. (1917)Ber. Saechss. Acad. der Wiss. Leipzig Math.-Phys. KI. 69,262-267 3 DeRosier, D. J. and Klug, A. (1968) Nature 217, 130-134 4 Crowther, R. A. (1971) Philos. Trans. R. Soc. London Ser. B. 261,221-230. 5 Henderson, R. and Unwin, P. N. T. (1975) Nature 257, 28-32 6 Crowther, R. A. and Klug, A. (1975) Annu. Rev. Biochem. 44,161-182 7 Hoppe, W. (1974) Naturwiss. 61,534-536 8 Dover, S. D., Elliot, A. and Kernaghan, A. K. J. (1981). Microscopy 122, 23-33 9 0 l i n s , D. E. et al. (1983) Science 220,498-500 10 Skoglund, U., Andersson, K., Strandberg, B. and Daneholt, B. (1986) Nature 319, 56(k-564 11 Daneholt, B. (1982) in Insect Ultrastructure (Vol. 1) (King, R. and Akai, M., eds), pp. 382-401, Plenum Press 12 Andersson, K., BjOrkroth, B. and Daneholt, B. (1980) Exptl Cell Res. 130,313-327 13 Olins, A. k., Olins, D. E. and Franke, W. W. (1980) Eur. J. ~ ' l l Biol. 22,714-723
For technical reasons we are unable to reproduce this figure in colour. See the December issue of Trends in Biochemical Sciences for full colour illustration.
T I B S 11 - D e c e m b e r 1986 14 Skoglund, U., Andersson, K., Bj6rkroth, B. and Daneholt, B. (1983) Cell 34, 847-855 15 Lamb, M. M. and Daneholt, B. (1979) Celll7, 835-848 16 Crowther, R. A., DeRosier, D. J. and Klug, A. (1970) Proc. R. Soc. London Ser. A 317,319340 17 Ottensmeyer, F. P. (1982) Science215, 461-466 18 Lewis, P. R. (1977) in Practical Methods in Electron Microscopy (Vol. 5) (Glauert, A. M., ed.), pp. 137-287, Elsevier 19 Williams, M. A. (1977) in Practical Methods' in Electron Microscopy (Vol. 6) (Glauert, A. M.,
503 ed.), pp. 1-76, Elsevier 20 Singer, S. J., Tokuyasu, K. T., Dutton, A. H. and Chen, W. T. (1982) in Electron Microscopy in Biology (Vol. 2) (Griffith, J. D., ed.), pp. 55-106, J. Wiley 21 Roth, J. (1983) in Techniques in lmmunocytochemistry (Vol. 2) (Bullock, G. R. and Petrusz, P., eds), pp. 217-284, Academic Press 22 Oakes, M. I., Clark, M. W., Henderson, E. and Lake, J. A. (1986) Proc. Natl Acad. Sci. USA 83,275-279 23 Somlyo, A. P., Somlyo, A. V., Schuman, H.
24 25 26 27 28
and Stewart, M. (1979) Scanning Electron Microsc. 2, 711-722 Cantino, M. E. and Hutchinson, T. E. (1982) Trends Biochem. Sci. 7,132-134 Crewe, A. V. (1983) Science, 221,325-330 Bazett-Jones, D. P. and Ottensmeyer, F. P. (1981) Science 211,169-170 Rowland, S. W. (1979) Topics Appl. Phys. 32, 9-79 Robb, R. A. (1982). Annu. Bey. Biophys. Bioeng. 11,177-201
499
Emerging Techniques Electron microscope tomography Ulf Skoglund and Bertil Daneholt Electron microscope tomography allows three-dimensional reconstruction of ultrastructural objects at the molecular level. The method is general and not limited to symmetric, or regularly ordered structures. Alone, or in combination with immunoelectron microscopy and electron spectroscopic imaging, electron microscope tomography is a powerful technique in cell and molecular biology.
Electron microscopy was a breakthrough technique in the analysis of the structure of the cell: cellular organelles already known could be described in a much greater detail, and new organelles and membrane systems were revealed. The subsequent ability to fractionate cells into well defined cellular components has made possible the biochemical study of cell structures, and electron microscopy is often used to verify the nature, quality and purity of a preparation. More recently, application of recombinant D N A technology and related techniques has allowed virtually any eukaryotic gene to be isolated and the corresponding protein prepared in large quantities and characterized. The main problem is usually to establish the function of a given protein, especially as the proteins are frequently components of complex cell structures. Thus, an important task for present-day biochemists is to reconstitute such complex structures from well defined molecular components and to elucidate the function of the constituents. The need for ultrastructural information, both in vivo and /n vitro, to match the development of the biochemical analysis of the cell becomes more and more obvious. Electron microscope tomography is a new technique that can be used to reconstruct ultrastructural objects in three dimensions at the molecular level.
Three-dimensional techniques A three-dimensional image of an electron microscopic specimen-can be obtained by inspecting two different electron micrograph projections of the U. Skogland and B. Daneholt are at the Department of Medical Cell Genetics, Medical Nobel Institute, Karolinska Institute, Box 60400, S-104 Ol Stockholm, Sweden.
object with a stereo viewer I. This procedure is often useful, but does not permit precise and quantitative descriptions of an object in three dimensions. Thus, there has been an urgent need to develop techniques for three-dimensional reconstruction of ultrastructural objects. An electron microscopic image is a two-dimensional projection of a threedimensional structure. By collecting a certain number of different projections of an object, one has sufficient information to accomplish a proper three-
\
i
,;,
dimensional reconstruction (Fig. 1). The mathematical theory for such a reconstruction approach was worked out as long ago as 1917 by J. Radon 2. DeRosier and Klug 3 were the first to apply this theory in electron microscopy when they reconstructed the spiral-shaped segment of phage T4 and made use of its helical symmetry to obtain a reliable reconstruction. The subsequent development of the mathematical and numerical aspects of the theory made it possible to study spherical virus particles 4 as well as molecular entities highly ordered in two dimensions s. These studies of symmetrical, or regularly arranged structures established the three-dimensional reconstruction method as a powerful tool for structural molecular research (for review, see Ref. 6). It is evident that a reconstruction procedure would have much greater applicability if the need to utilize symmetries in the analysis could be overcome. The paramount problem with the analysis of non-symmetric structures is that the different projections are difficult to align precisely with each other (high precision in defining a common origin is crucial for the final resolution of the reconstruction). An early aligning approach was based on the calculation of
I
,.,
" i v
Fig. 1. The principle of three-dimensional image reconstruction from electron micrographs. Five micrographs, shown in a semicircular arrangement, represent a set of projections of a centrally placed specimen which are accurately aligned in relation to each other to allow image reconstruction. The three-dimensional reconstruction takes place as a series of two-dimensional reconstructions: in each single step, one of which is shown in the figure, a circular disc is reconstructed by back-projection (after suitable weighting) of the corresponding lines in the micrographs e7,2~. Note that the lines and the reconstructed disc are perpendicular to the tilt axis. Several consecutive discs ultimately form a cylinder in which the reconstructed object is contained. Reproduced, with permission, from Ref. 10. ~) 1986,ElsevierSciencePublishersB.V.. Amsterdam 0376 5067/86/$02.00
500 cross-correlation functions between successive tilts 7. More recently, colloidal gold particles spread on thin sections have been used as reference points for alignment procedures 8.9. These early studies showed the feasibility of reconstructing an aperiodic object from electron micrographs in a tilt series. Our laboratory has recently worked out a new method for the three-dimensional reconstruction of ultrastructural objects m, symmetric and asymmetric, to a resolution better than 50 A. The origin problem has been solved by the colloidal gold method: the coordinates of small gold particles are determined to calculate image beam rotation, tilt angle, shrinkage, changes of planarity and parallaxes in the whole tilt (projection) series. A coherent computer software package has been put together, tested and is now used routinely. The technique has been applied to ultrathin sections for studies of nucleic acid-protein complexes at molecular dimensions. To demonstrate the power of the method and the nature of the results we will discuss a recent investigation of a specific premessenger ribonucleoprotein (RNP) particle 10.
Experimental material Giant polytene chromosomes in the salivary glands of the midge C h i r o n o m u s tentans were chosen for ultrastructural studies (for review, see Ref. 11). These chromosomes have acquired their exceptional size by juxtaposition of thousands of identical elements, each equivalent to a chromosome in a diploid cell. The chromosomes can be identified easily and specific chromosome regions investigated with electron microscopy. We have concentrated our studies on two selected segments of chromosome IV, the Balbiani rings 1 and 2. These chromosome segments harbour genes coding for large secretory proteins. The Balbiani ring (BR) genes are transcriptionally very active in the salivary glands and have been characterized by electron microscopy both in situ 12-14 and after chromosome IV has been isolated and spread on a surface 15. It has been possible to observe how the large primary transcripts (75S RNA) are synthesized in the BRs andpacked together with proteins into 500 A diameter RNP particles. The abundance and exceptional size of BR particles makes them readily identifiable in the nuclear sap and in the nuclear pores. We have used the tomography method to reconstruct the BR particle in three dimensions during its synthesis, its transport to the nuclear pore and its passage through the pore.
T I B S 11 - D e c e m b e r 1986
Computed tomography technique Ultrathin sections through salivary glands were prepared by conventional procedures, and colloidal gold particles were deposited onto the sections to obtain alignment markers. The specimens were examined in a conventional transmission electron microscope provided with a goniometer to allow tilting. A suitable nuclear area with BR particles was identified and photographed as a series of tilted views (every 10° in the range +60 °) (Fig. 2). Prior to the actual threedimensional reconstruction the electron micrographs were digitized and aligned with an iterative full least-squares procedure using the gold particles as alignment markers (G. Bricogne and U. Skoglund, unpublished). Information was selected from each electron micrograph for the reconstruction (Fig. 3). The three-dimensional reconstruction followed the 'filtered back-projection principle', in which the Fourier transforms of the projections (tilts) are radially weighted 16. As shown in Fig. 1, each three-dimensional reconstruction was composed of a series of two-dimensional reconstructions of circular disks perpendicular to, and repeated along, the tilt axis, forming a cylinder. The completed reconstruction was shown as a number of density maps corresponding to the reconstructed sections through the object. The particle as a whole was presented both as a balsa-wood model (Fig. 3 in Ref. 10) and as a contoured vector image on a display (see Fig. 4).
Three-dimensional structure of the BR RNP particle Four individual BR RNP particles were reconstructed: they displayed strikingly similar gross morphology irrespective of their orientation within the sections. The high correlation coefficients between the various particles (about 0.8) permitted the calculation of an average structure. This average particle is presented in Fig. 4: it has a hole in the centre and a slit at 1-2 o'clock, giving the particle a ring-like appearance. The RNP element is asymmetric and can be described as a thick ribbon divided into four separate domains, designated 1-4 (Fig. 4A). Furthermore, the asymmetry of the particle made it possible to predict the location of the two ends of the R N A molecule. By comparing the features of the growing RNP particle on the gene and the domain organization of the particle, it was concluded that domain 1 is completed first and probably contains the 5' end of the R N A molecule, while domain 4 is the last one to be finished and therefore probably harbours the 3' end. The ring-like structure with the two ends of the R N A molecule close together in the particle can have important functional implications for the splicing, polyadenylation and transport processes (for discussion, see Ref. 10). Resolution of the method The BR RNP particles were reconstructed to a resolution of 80-90 ~ , which is close to the predicted theoretical
!
Fig. 2. Electron micrograph of Balbiani ring R N P particles in the nucleus of a salivary gland cell. The nuclear envelope separates nuclear sap (left) from cytoplasm (right). The B R R N P particles in nuclear sap are encircled, and a large arrow denotes a BR particle being translocated through a nuclear pore. Some of the gold particles serving as alignment markers are indicated by small arrows. The photograph represents a 0 ~ degree tilt in a tilt series comprising 13 photographs covering the --60~ range. The scale bar corresponds to 2000 A. For experimental details, see Ref. 10.
T I B S 11 - D e c e m b e r
501
1986
well as stain densities; separate reconstructions can be compared and differences and similarities evaluated; average structures can be calculated and interpreted; selected segments of a structure can be studied separately and compared with other segments or with other particles to find similar, or complementary, structures; specific elements within a structure can be traced. It is also possible to construct specific models and compare those with the structure obtained. It should be stressed that m o d e m computer graphic techniques offer excellent aids for this type of analysis.
Fig. 3. Thirteen extracts from a tilt series displaying the same B R R N P particle in the centre. Together these extracts represent an aligned set of projected views of the particle and are used as input data for the reconstruction algorithm. The tilt axis runs vertically through the middle o f each extract; the tilt angles have been indicated with labels. A few colloidal gold markers are visible as dark circular objects. Every extract is 3100 fii square. The image was produced on a display and photographed directly on the screen.
limit (85 A.) for a 500 A_diameter object, photographed under optimal conditions in 10° intervals in the range _+90° (Ref. 16). This result suggests that the restriction of tilt angles to _+60° on m o d e m electron microscope goniometers only has a minor effect on the final structure when the object has good contrast. The key to a high resolution in the tomographic procedure is the proper alignment of the electron micrographs comprising the tilt series. For our routine reconstructions today we generally achieve a 6 A. prediction error for any point anywhere in the tilt series. This allows us to get high quality reconstructions of the 500 A particles at a resolution better than 50 A when the tilt series comprises 25 electron micrographs taken at 5 ° intervals between _+60° . It should be noted that smaller objects than the B R particle will be reconstructed at a still higher resolution, because the resolution in a given reconstruction is inversely proportional to the thickness of the stained object 16. Since there is no indication of radiation damage, it seems likely that we can further improve the resolution by increasing the
number of tilted views of the specimen (the resolution is directly proportional to the number of evenly distributed tiltsl6). The ultimate limit for the resolution in a positively stained specimen is set by the stain distribution and has been estimated to be about 20 ,~ (Ref. 17). It is not yet possible to predict how close to this limit the computed tomography can get.
Analytical power In conventional transmission electron microscopy it is often difficult to demarcate a specific structure because of the surrounding background or superimposed structures. These problems are usually circumvented by the threedimensional reconstruction method. Furthermore, in tomography the reconstructed object can be rotated and studied from all directions, not only from those available in the electron microscope. The most essential point is, however, that the tomography approach permits a precise quantitative description and advanced analysis of the object. A given reconstruction can be studied in a number of quantitative ways: volumes and distances can be measured as
Combination of computed tomography with other methods Electron microscope tomography will be most useful when it is combined with methods that provide information on the biochemical nature of the structure. Specific enzyme activities can be detected by local deposition of electron-dense material on the structure (enzyme cytochemistry) TM. A more general and precise approach would be to use specific ligands tagged with electron-dense markers, preferably colloidal gold, in order to locate defined macromolecules at the ultrastructural levellg-2k Antibodies, ideally monoclonals, represent the most obvious and versatile possibility (immunoelectron microscopy), but there are a rapidly increasing number of other ligands available: hormones for their receptors, lectins for specific moieties of glycoproteins and glycolipids, nucleases for their nucleic acid substrates, low density lipoproteins for their membrane receptors, etc. For nucleic acid-containing structures, in situ hybridization could also be an attractive possibility; a recent study of ribosome structure has shown that a short, single-stranded D N A probe labelled with biotin can be hybridized to the corresponding ribosomal R N A sequence and visualized in the electron microscope after addition of avidin 2:. An elemental analysis of the ultrastructural object could be useful in some circumstances. The conventional X-ray microanalysis can provide information on atoms 23 but usually the resolution is too low to be optimal for molecular entities (spot anal)zsis can reach a resolution of about 200 A; Ref. 24). For biological objects the new approach of using scattered electrons to determine the energy loss spectrum (electron energy loss spectroscopy) seems more promisingS7.25; electron spectroscopic imaging is particularly sensitive, with a more than tenfold better resolution than X-ray microanalysis iT. One example of the
502
T I B S 11 - D e c e m b e r 1986
Fig. 4. The average B R R N P structure represented as a three-dimensional vector model. Six views are shown, labelled A - F . Four domains are outlined in A and numbered 1~l; the red line (upper righO demarcates the slit between domain 1 and domain 4. A s a further aid to viewing B-E, the demarcation line and the positions o f domain 1 and 4 are shown. Domain 2 constitutes the major part o f the bottom view o f the structure seen in F. The image was composed and photographed on a
display.
feasibility of the electron spectroscopic imaging method at the molecular level is the elucidation of the path of D N A around the histone octamer of a nucleosome by analysis of the phosphorus distribution in individual nucleosomes 26.
Applicability We have stressed that electron microscope tomography allows the reconstruction of any transparent ultrastructural object in three dimensions. One example has been given from our own research programme, but it is clear that the method has a very wide spectrum of applications in cellular and molecular biology. It should be possible to analyse a variety of subcellular organelles, filamentous structures and large molecular complexes such as ribosomes. The method should be well suited to study dynamic processes such as intracellular transport, receptor-ligand interactions of various kinds and cell-to-ceU contacts.
Computed tomography in itself will provide the distribution of mass in the molecular complexes, and when combined with the use of specific ligands coupled to electron-dense markers it will be a most powerful tool to identify and localize specific macromolecules within the cell. It seems likely that electron microscope tomography will contribute effectively to change the emphasis in cell biology from ultrastructural objects to molecular entities.
Acknowledgements We are grateful to Hans Mehlin and Anna Sj6d6n for stimulating discussions and technical assistance and to Evy Vesterb~ick for typing the manuscript. This research was supported by Knut and Alice Wallenberg Foundation, Swedish Natural Science Research Council and National Swedish Board for Technical Development.
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For technical reasons we are unable to reproduce this figure in colour. See the December issue of Trends in Biochemical Sciences for full colour illustration.
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