High resolution cryo electron microscopy of specifically stained specimens

High resolution cryo electron microscopy of specifically stained specimens

Ultramicroscopy 15 (1984) 205-214 North-Holland, Amsterdam 205 H I G H R E S O L U T I O N CRYO E L E C T R O N M I C R O S C O P Y OF S P E C I F I...

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Ultramicroscopy 15 (1984) 205-214 North-Holland, Amsterdam

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H I G H R E S O L U T I O N CRYO E L E C T R O N M I C R O S C O P Y OF S P E C I F I C A L L Y S T A I N E D SPECIMENS E. K N A P E K , G. L E F R A N C and I. D I E T R I C H Siemens AG, Research and Development, P.O. Box 830952, D-8000 M~nchen 83, Fed. Rep. of Germany

and H. F O R M A N E K Botanisches lnstitut der Universti~t Mi'mchen, Menzingerstrasse 67, D-8000 M~nehen 19, Fed. Rep. of German),' Received 2 March 1984 Dedicated to Professor Benjamin Siegel on the occasion of his 68th birthday

At very low temperatures organic specimens with attached metal atoms can be irradiated with a relatively high electron fluence without losing the characteristic features of their architecture. This is demonstrated on three examples: a metal-organic molecule, a membrane-like biological layer, and a three-dimensional polymeric copper complex.

1. Introduction

A few electron microscopes with superconducting lenses have been in operation for several years [1 3]. Since the electron-optical quality and the electro-magnetic and mechanical stability of the instruments is high, a resolution below 0.2 nm can be obtained. In principle, high resolution imaging of "cold" specimens is no problem. If, however, we take organic, particularly biological, objects the crux is the radiation damage. In the last years some experience has been acquired on how the electron impact affects the high resolution imaging of organic material kept at low temperature. A short survey on the conclusions we can now draw is given as an introduction. First we consider native, i.e. unstained, periodically ordered specimens. The radiation damage was investigated by electron diffraction. Direct high resolution imaging also gave hints in this direction. The results on the so-called cryoprotection against radiation, i.e. the improvement in structure conservation due to cooling, varied widely [4,5]. We think we are now able to explain at least

partly the reasons for the differences, Since in the past the diffraction method was mainly used at room temperature for studying the decrease in crystallinity due to electron radiation, it has also been applied for the low temperature measurements. One should, however, keep in mind that this method is not especially suitable for a comparison of measurements carried out on different instruments. Many parameters with errors of at least 20% are involved so that easily deviations of a factor two can be expected. But the main point is the role of the specimen preparation for the low temperature investigations. The electrical and the thermal conductivities of most organic materials are extremely low, when the temperature approaches 0 K, so that charging and heating of the specimen is much more dangerous than at room temperature. In addition, the contact between the object and the carrier, e.g. a carbon foil, necessary for the energy dissipation may weaken due to cooling. This effect is caused by different thermal contraction coefficients of the different materials and the strong tensions produced as a consequence, so that the original con-

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E. Knapek et al. / HR crvo EM of spec(ficallv stained specimens

tact may even break. In this case the object may drift and tilt on the carrier in the beam due to the forces caused by charging. This is of influence to the intensity of the reflections in the diffractogram which may vary at random. There is, however, no connection to a change in crystallinity. The test experiments are described elsewhere in more detail [6]. If the specimens were prepared so that the possibility for discharging and the mechanical stability (e.g. by embedding) were guaranteed, a structure conservation took place as a consequence of the cooling of the specimen. The results of direct imaging showed the same trend. In case the preparation was under control, the resolution of some micrographs exposed to a fluence of the order of 1000 e / n m 2 with a beam voltage of 100 kV was in favorable cases below 0.5 nm, a value never obtained at room temperature [1,8]. So there is no doubt that cooling of the specimen reduces the radiation damage, if a suitable preparation is used. In case negative stain is applied to the specimen, the radiation damage is reduced at room temperature but the resolution is limited due to clusters of heavy metal atoms. If the specimen is cooled to low temperatures the damage produced by electron impact is stronger than at room temperature, even if specimen carriers with reasonable conductivities are chosen. This was the result of a number of up to now unpublished investigations on material such as HPI layer, ribosomes, tobacco mosaic virus and a membrane protein from urinary bladders. We explain these results by a damage which already occurs during cooling. The different thermal contraction coefficients produce tensions which may even cause cracks. The ensuing radiation enhances the effect. On specifically stained specimens, i.e. material where the metallic component is chemically attached to a well defined location of the molecule, a high structure conservation under the beam was observed at low temperatures, which permitted high resolution imaging in every case, The results were confirmed by recent studies of Lamvik et al. [9] on a biological membrane specifically stained with Hg, where the electron diffraction method was applied. The advantages of the

"specific stain" for high resolution low temperature electron microscopy are discussed on micrographs of three organic substances: a metal-organic molecule, a specifically stained membrane and a polymeric copper complex.

2. Hexaphenylen mercury The micrographs were taken some time ago [10] when experience on imaging of organic material with superconducting lenses was still limited. We repeat here partly the description of the imaging procedure. A small amount of the molecules (fig. 1), less than required for a monomolecular layer, was spread on an - 3 nm thick amorphous aluminum oxide film which was supported by a holey carbon foil. The electron fluence for imaging was high. For focusing and correcting the astigmatism a fluence of the order of 10 ~ e / n m 2 was required and for exposing a single micrograph (magnification 400,000: 1) the fluence amounted to - 105 e / n m 2. Several focus series with 8 to 10 micrographs were taken. The noise of the aluminum oxide film was by far lower than that of the support film, but in spite of the low background noise a single mercury atom could not have been detected in bright field, as can be easily calculated [11]. In our case, however, where the mercury atoms are arranged in a hexagon (fig. 1), the contrast was considerably enhanced due to the symmetry properties. In fact many hexagonal structures could be detected on several focus series, if the aluminum oxide film was sufficiently thin (fig. 2). Keeping in mind that noise of the photographic emulsion, quantum noise

Fig. 1. Structure model of the hexaphenylen mercury molecule.

E. Knapek et al. / HR co, o EM of specifically stained specimens

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E. Knapek et al. / HR crvo E M of specifically stained specimens

Fig. 2, Micrographs of hexaphenylen mercury. Beam voltage 215 kV, electron optical magnification 500,000: 1, accumulated electron exposure 1.5 × 106: (a) and (b) belong to the same focus series; defocus (generalized) 1 and 1.5 respectively; (c) was taken from a different focus series.,

and noise of the aluminum oxide film are superposed the hexagons stand out remarkably well. Structures of this kind were not found on thicker aluminum oxide films and on the carbon support films. Further indications that the hexagonal structures one can recognize consist of mercury atoms are given in ref. [10], e.g. a comparison of the intensity distribution in the light optical diffractogram and the calculated phase contrast transfer function. In comparing the micrographs, figs. 2a and 2b, taken one after the other in the same series with normalized defocus distance 1 and 1.5, the following features attract attention. There is quite a difference in the patterns on the carbon foil in contrast to those on aluminum oxide where the pattern gives the same main impression. The latter statement does not hold for details below 0.4 nm. This can be easily explained since according to the phase contrast transfer function phase reversal starts on fig. 2a for periodicities of this order. The encircled ring structures, however, do not change much with one exception, though their periodicities are near to the gap of the phase contrast transfer function. This can be explained by scattering contrast which shows no phase reversal and which contributes to the contrast of symmetrically ordered mercury atoms. The ring num-

ber 5 is apparently produced by phase contrast only and does not consist of mercury atoms. The image fig. 2c, which also shows several more or less hexagonal structures, is taken from a different focus series. According to the attached light optical diffractogram the 0.35 nm periodicity nearly coincides with a gap in the phase contrast transfer function. We think it is plausible that at room temperature the molecules are destroyed in the beam. The mercury atoms diffuse along the surface and form clusters or evaporate. As a consequence of cooling the specimen the mercury atoms are fixed to the carrier film, and even if the benzene rings should be damaged, the mercury rings do not break.

3. Murein Another example for the statement that heavy atoms keep their position in specimens at low temperatures in spite of a strong electron beam is murein or peptidoglycan, a membrane-like structure. Murein is the rigid layer of the bacterium cells walls, a derivative of chitin. It consists of a network structure (fig. 3) of N-acetulmuramic acid (M) and N-acetylglucosamine (G). Perpendicular to the network peptide chains stick out, symbolized as vertical lines. About 50% cross-linking

E. Knapek et al. / HR co'o E M of specifically stained specimens

--~G--M--G--M"'-~

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Fig. 3. Network structure of murein. G: N-acetylglocusamine; M: N-acetyl muramic acid; Z: cross-linking by hydrogen bonds; e : Pt atoms attached for marking.

takes place between the free amino group on one peptide chain and the terminal carboxylgroup of the neighboring chain. After incorporating sulfhydryl groups the remaining free amino groups could be marked with Pt atoms due to a reaction with Pt blue as described in ref. [12]. According to the rough sketch of the structure (fig. 3), we should observe lattice lines of - 0 . 5 and - 1 nm distance in the electron micrographs if the murein layer was not completely destroyed by radiation during the exposure. There is a further indication that periodicities of the order of 0.5 and 1 nm should be characteristic for murein. The reflections with the highest intensities according to the existing structure models can be calculated by a Fourier transform [13]. According to these calculations the reflection with by far the highest intensity should belong to the packing periodicity of the peptidoglycan strands (0.45 nm). A further reflection which has also been detected in X-ray Debye-Scherrer patterns is caused by the periodicity of G M peptide units ( - 1 nm) and should be oriented more or less perpendicular to that of the packing periodicity. The preparation of murein is described in refs. [12,14]. The specifically stained layers in the shape of sacculi are spread on top of graphite oxide crystals ( - 2 nm thick) which are supported by a holey carbon film. The contrast of the murein sacculi labelled with Pt (twofold thickness of one layer - 4 nm) is sufficiently high for distinguishing them from the background in conventional microscopes. High resolution imaging, however, was not successful.

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Hence at very low temperature several image series were taken, with a very high fluence, which exhibit the 0.5 nm lattice lines of murein [15]. There were some doubts at that time whether artefacts were observed particularly since no angle variations and no other lattice distances were found. In the meantime most of the micrographs on which murein should be imaged were studied by scanning over the micrographs with a laser beam, and light optical diffractograms were taken from small areas. Besides reflections belonging to lattice distances between 0.45 and 0.5 nm, those produced by - 1 nm lines were frequently found. An example is the light optical diffractogram fig. 4b taken from the section shown in fig. 4a. One can also recognize the mentioned lattice lines in the micrograph. The angles are slightly tilted against the main direction on several spots. This can be seen more clearly on fig. 4c, where the lattice lines outlined in fig. 4a are traced. Apart from the larger crystalline area the micrograph as a whole gives the same impression as those of such " a m o r p h o u s " inorganic materials as TaSi [16], where small crystallites are embedded in a matrix of at-random arranged atoms. The murein image indicates that even with a fluence of 3 × l0 s e / n m 2 the main features of the structure are preserved and the dynamic in this biological material is visualized. But it seems to be necessary to attach heavy atoms to the specimen and to keep it at very low temperatures during irradiation.

4. Copper c o m p l e x of B S H

High resolution imaging of the three-dimensional polymer [Cu 2 BSH]., the copper complex of N,N'-bis-salicyloyl-hydrazine, could only be carried out at very low temperature since at room temperature the fluence which started to damage the crystallinity was at least two orders too low for taking micrographs with sufficient contrast. Images such as fig. 5, which show periodicities of - 1 nm in spite of irradiation with a fluence of - 4 × 104 e / n m 2 were published before, particularly in connection with the structure determination of the complex [17]. In this paper we try to explain why

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C J J J J

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Fig. 4. Electron microscopy of murein. (a) Micrograph of murcin. Beam voltage 220 kV, accumulated fluencc 3.5 X 1 0 ~ e / n' n l - dcfocu~ 525 nm, electron optical magnification g4,000: 1. (b) Light optical diffractogram. (c) Tracing diagram of the lattice lines in micrograph (a). Some spots arc marked with arrows for correlation.

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Fig. 5. Micrograph with lattice lines of BSH. Beam voltage 220 kV, accumulated fluence 3.5×104 e/nm 2. electron optical magnification 80,000 : 1.

this structure is relatively radiation resistant at low temperature. In the model of [Cu 2 BSH] n one assumes that two BSH molecules are connected via one Cu atom, i.e. each Cu atom is linked with its four coordination positions to two BSH molecules (fig. 6). Microcrystals of the complex in a suitable size for electron microscopy could only be obtained if a m m o n i a was added to the solution from which the crystals precipitate. This can be explained since according to model calculations (not yet published) the angles between the BSH molecules have a certain range of variation in the energy minimum, and for this reason the complex without a m m o n i a is nearly amorphous. If a m m o n i a dif-

fuses into the complex it forms a relatively weak fifth coordination b o n d to each Cu atom. As a consequence the angles of the attached four BSH molecules are fixed due to the space requirements of N H 3. Crystals can grow only under such conditions. This means that actually the imaging was performed on the complex [ C u 2 ( N H 3 ) 2 B S H ] , . Since the a m m o n i a can easily be removed by heating the material in vacuo one can conclude that exposing the crystals to an electron beam may result in a volatilization of the ammonia. As a consequence the crystallinity should be destroyed, and this really happens by a fluence below 100 e / n m 2 as proven by electron diffraction. At liquid helium temperature there is no detectable mass

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Fig. 6. Model of tetramers of the copper BSH complex.

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Fig. 7. "Three-dimensional" model of the copper BSH complex. Three layers of tetramers each containing 10 units give an implesslon of the three-dimensional c o n s t r u c t i o n , where the 1 nm periodicity is especially prominent.

E. Knapek et al. / HR c~o E M of specifically stained specimens

loss due to the electron b e a m , i.e. besides others the a m m o n i a molecules are t r a p p e d . Even if ionization of p a r t s of the BSH molecule should b r i n g a b o u t r a d i a t i o n d a m a g e , which might be detectable, e.g. b y electron energy loss spectroscopy, the m a i n skeleton of the structure r e m a i n s intact. This is especially caused by the stabilizing effect of copper. T h a t the lattice lines should still be visible even if smaller d i s a r r a n g e m e n t s of the C, O or N a t o m s take place can be derived from the F o u r i e r t r a n s f o r m of the structure model. W i t h o u t calculations, the strong periodicity can be recognized on the m o d e l lattice which corres p o n d s to a 2.5 n m thick crystal (fig. 7). The r a d i a t i o n resistance is also c o n f i r m e d b y electron diffraction, since even h i g h - o r d e r reflections belonging to periodicities of 0.06 n m are still visible after i r r a d i a t i o n with 3.5 × 107 e / n m 2 at a b e a m voltage of 220 kV [17].

5. Conclusions The first example, the imaging of the organic m e r c u r y c o m p o u n d , m a k e s it evident that the heavy a t o m s stick to the s u p p o r t layer, even if there is a s t r o n g electron i m p a c t in case the object t e m p e r a ture is sufficiently low. The second example, the murein, d e m o n s t r a t e s that due to the fixed position of the metal a t o m s the crystallinity is conserved at low temperature, though we must assume that only a skeleton of the biological material c o u l d have survived such a high electron exposure. T h e same is true for the third example. F u r t h e r more, in this case a c o m p o n e n t of the p o l y m e r

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necessary for the crystallization is volatile at r o o m t e m p e r a t u r e u n d e r the beam. A t low t e m p e r a t u r e this c o m p o n e n t c a n n o t be removed, so that the c r y o p r o t e c t i o n is p a r t i c u l a r l y impressive.

References [1] I. Dietrich, F. Fox, E. Knapek, G. Lefranc, K. Nachtrieb, R. Weyl and H. Zerbst, Ultramicroscopy 2 (1977) 241. [2] G. Lefranc, K.-H. Mi~llerand I. Dietrich, Ultramicroscopy 6 (198l) 291. [3] M.K. Lamvik, R.E. Worsham, D.A. Kopf and J.D. Rohertson, Ultramicroscopy 12 (1983) 79. [41 E. Knapek and J. Dubochet, J. Mol. Biol. 141 (1980) 147. [5] J. Lepault, J. Dubochet, I. Dietrich, E. Knapek and E. Zeitler J. Mol Biol. 163 (1983) 511. [6] E. Knapek, H. Formanek, G. Lefranc and I. Dietrich, in: Proc. European Congr. on Electron Microscopy, Budapest, 1984, Vol. II, p. 1395. [7] F. Zemlin, E. Beckmann, E. Reuber, D. Dorset and E. Knapek, in: Proc. European Congr. on Electron Microscopy, Budapest, Vol. I, p. 241. [8] W. Chiu, T.W. Jeng, F. Zemlin et al., in: Proc. European Congr. on Electron Microscopy, Budapest, 1984, Vol. II, p. 1483. [9] M.K. Lamvik, D.A. Kopf and J.D. Robertson, Nature 301 (1983) 332. [10] H. Formanek and E. Knapek, Ultramicroscopy 4 (1979) 77. [11] J. Frank, Biophys, J. 12 (1972) 484. [12] H. Formanek and S. Formanek, European J. Biochem. 17 (1970) 78. [13] H. Formanek, Z. Naturforsch. 37c (1981) 226. [14] H. Formanek, Ultramicroscopy 4 (1979) 227. [15] I. Dietrich, H. Formanek~ F. Fox. E. Knapek and R. Weyl, Nature 277 (1979) 380. [16] K. Schober and E. Knapek, in: Proc. European Congr. on Electron Microscopy, Budapest, 1984, Vol. II, p. 1253. [17] I. Dietrich, H. Formanek, W. von Gentzkow and E. Knapek, Ultramicroscopy 9 (1982) 75.