Electron diffraction studies of membranes

Biochimica et Biophysica Acta, 472 (1977) 345-371 © Elsevier/North-Holland Biomedical Press B B A 85175

ELECTRON

DIFFRACTION

STUDIES

OF

MEMBRANES

S. W. H U I

Electron Optics Laboratory, Biophysics Department, Roswell Park Memorial Institute, Buffalo, N.Y. 14263 (U.S.A.) (Received F e b r u a r y 25th, 1977)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

345

II.

Theoretical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . .

347

A. Scattering o f electrons by m e m b r a n e molecules B. Structural analysis . . . . . . . . . . . . . C. L i m i t a t i o n s . . . . . . . . . . . . . . . 1. B e a m coherence . . . . . . . . . . . . . 2. Selected area . . . . . . . . . . . . . . . 3. Specimen irregularity . . . . . . . . . . . 4. C a m e r a length . . . . . . . . . . . . . .

347 349 351 351 351 352 352

III.

IV.

Specimen preservation m e t h o d s

. . . . . . . . . . . . . . . . . . . . . . . .

352

A. D e h y d r a t i o n effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Air or v a c u u m drying . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Sugar substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. E n v i r o n m e n t a l c h a m b e r . . . . . . . . . . . . . . . . . . . . . . . . . B. R a d i a t i o n effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

353 353 353 353 353 354

Results A. B. C. D.

V.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Electron diffraction f r o m m e m b r a n e lipids . . . . . . Electron diffraction f r o m m e m b r a n e proteins . . . . . Electron diffraction of cell m e m b r a n e s . . . . . . . . Kinetic studies . . . . . . . . . . . . . . . . . . .

Conclusion

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

356

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356 363 366 367

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

367

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

368

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

369

Acknowledgements References

. . . . . . . . . . . .

I. I N T R O D U C T I O N Diffraction fortunately, periodic

most

in nature.

methods

are

of the known

most

useful

molecular

in studying

arrangements

Thus, in order to study the molecular

periodic

structures.

in biomembranes architecture

Un-

are non-

of membranes,

346 one often has to resort to the techniques of imaging rather than to diffraction. However, direct electron imaging of the macromolecules in biomembranes is hampered by the lack of contrast from the native molecules, as well as by the damaging effects oftheincident electron beam. Electron imaging using stained, sectioned, or replicated specimens are limited in resolution, and the results require careful interpretation. These methods also preclude any direct physico-chemical measurements on membranes comparable to X-ray data. Diffraction from well-preserved natural molecular arrays, on the other hand, can provide structural information accurate to fractions of an angstrom. The signal-to-noise ratio is improved over corresponding electron images due to the pooling of redundant information from repeating units. Therefore, although there exist only a limited number of naturally-occuring repeating structures in membranes, a great deal of effort has been spent in their studies. In cases when natural arrays are unavailable, artificially stacked membranes have been used to create periodical structures for diffraction studies. This paper is concerned mainly with the progress in applying electron diffraction techniques to obtain molecular information from 'intact' membranes. The membrane structures as revealed by staining or replication techniques have also been studied by electron diffraction or by optical diffraction from their corresponding micrographs. These types of studies mainly served as an aid to electron microscopy, and thus will not be covered by this paper except in cases related to the 'intact' membrane studies. In the last fifty years since the discovery of electron diffraction by Davisson and Germer [1], the technique has been widely adapted to the structural analysis of materials [2]. Yet, its application in the analysis of biomembrane structure has been scant in comparison to the techniques of X-ray [3] and neutron diffraction [4]. The main difficulties in the use of electron diffraction in the structural analysis of biological materials are the radiation damage to the specimen by the electron beam and the requirement that the specimen be placed in a vacuum for electron diffraction experiments. Both requirements are destructive to the delicate biological molecules to be studied. As technology develops, these difficulties gradually become less insurmountable, and meaningful results have been obtained from electron diffraction studies. The advantages of electron diffraction in membrane studies are gradually being recognized. The two main advantages of electron diffraction over other diffraction methods are the ability to obtain diffraction information from very small areas by the selective area diffraction technique, and the ability to obtain imaging information from the same specimen [5]. Both abilities rely on the fact that the electron beam, like any other charged particle beam, can be focused precisely. With selective area diffraction, an area of width as small as 20 nm can be singled out for study with the use of a scanning transmission instrument using a field emission source; therefore, localized structure information may be obtained. The ability to obtain an image from the same diffraction specimen not only reveals the morphology of the specimen, but also helps to solve the phases of certain diffraction intensities within the resolution of the image

347 TABLE I COMPARISON OF ELECTRON, X-RAY AND NEUTRON DIFFRACTION METHODS

Electron X-ray Neutron

Wavelength (A)

Typical atomic scattering amplitude (era)

Typical specimen thickness(ram)

0.01-0.05 0.7-2.3 1.5

10-8 10-11 10-12

10-5-10-4 10-1_1 1 -10

(see ref. 19). In these respects, electron diffraction is unmatched by most other diffraction techniques. In addition to these mentioned abilities, electron diffraction has many more advantages over other diffraction methods [5]: 1. Much shorter wavelength than that of the commonly used X-rays and thermal neutrons (see Table I). The short wavelength of the incident beam extends the limit of the resolution capability, although this extension may not be relevant in studying presently known biological structures. 2. Variable camera length adjustment ranging from less than a meter to hundreds of meters in one instrument - the electron microscope. 3. A strong specimen-beam interaction which enables in-plane structural information to be derived from a single layer of membrane, thereby avoiding the spatial averaging of information caused by superimposing multilayer membranes. 4. The shallow penetration of the electron beam, which is a hinderance in obtaining structure information of membrane profiles, may be utilized to selectively study the surface of the specimen by reflection diffraction. 5. The requirement of only a small quantity of specimen. This is particularly important in low yield preparations. 6. Extremely short time required to record information. This enables membrane dynamics to be observed without temporal averaging of information due to prolonged exposure. Therefore, in spite of the technical drawbacks, some effort has been made to utilize electron diffraction as a tool to study biomembrane structures. The remainder of this paper will review the exploratory efforts.

II. THEORETICAL CONSIDERATIONS

IIA. Scattering of electrons by membrane molecules The scattering of X-rays is related to the electron density 9-. In the same manner, the scattering of electrons is related to the atomic potential tp, which is determined by the nuclear charge 9+ as well as the electron density distribution 9- of the atom in the Poisson equation:

348 V 2 ~0= 4=e (9+ ÷ O-)

(1)

The atomic scattering amplitude for electrons,fe, and the atom scattering factors for X-rays, fx (in electron units), are thereby related by the Mott equation [6]: f~(s) - k ( Z - -

f.) / s z

(2)

where s = sin(0/2) is a reciprocal space vector, k is a constant, and Z is the atomic number. This relationship enables the well-developed X-ray scattering data to be applied to electron diffraction work within the kinematical limits. The ratio of f ~ / f x is much higher if high Z elements rather than the common elements in biological materials are involved (see tables in ref. 2). Nevertheless, for carbon in the scattering range of 1 nm -~, atomic scattering amplitude for electrons is approx. 10 -8 cm, whereas that for X-ray scattering is approx. 10-11 cm. The difference is due to the nature of scattering of these waves [2]. A comparison of electron, X-ray and neutron scatterings is presented in Table I . The strong interaction between the electron beam and the specimen gives many distinct features of electron diffraction. The thickness of the specimen coherently sampled by the electron beam is very limited. It is usually less than several hundred nanometers. The strongly scattered beam dramatizes the dynamical diffraction effects [7]. Since biological membranes are usually less than several tens of nanometers in thickness and their regular structures do not in general extend to the third dimension, these special considerations do not seriously hinder the utilization of the electron diffraction techniques in membrane structure studies. In contrast, because of the strong beam-specimen interaction, electron diffraction may be used to obtain localized structural information from just a single sheet of membrane. This very feature also dictates the geometry of the membrane specimen to be studied. Since meaningful diffraction information can be obtainable only if the specimen is less than several hundred nanometers thick, the specimens that give the best signal-tonoise ratio are those membrane specimens having their surfaces normal to the beam direction. Large and flattened vesicles resembling a flat double layer are also suitable for electron diffraction study. Vesicles filled with liquid have a poor signal-tobackground ratio. Because of the limited length ( ~ 100 nm) of coherent scattering, diffraction by tangential incidence to the membrane plane is restricted to membrane stacks less than several micrometers wide even with the use of high-voltage electron microscopes. For this reason, electron diffraction is more suitable for studying the structures in the plane of the membranes than the cross-sectional profile of the membranes. Eqn. 2 also gives the contrast limit for imaging and diffraction. The electron scattering at a fixed angle by single atoms is proportional to the atomic number. The total scattering of materials, or groups of atoms, is therefore proportional to the mass thickness of the material. For biomembranes, the thickness variation along the membrane is often small. The innate contrast is generally provided by the density variation of different areas or of different molecules on the plane of the membrane. A simple calculation shows that the mass thickness difference resulting from a typical spherical

349 protein molecule completely embedded in a phospholipid bilayer provides a contrast of 25 ~o from the lipid background. With the superimposing layer of the polysaccharide coat, this contrast could be even less. For this reason, unprocessed electron images of unstained membranes are often featureless. So far we have considered only the elastically scattered electrons, i.e., those electrons which do not impart energy to the specimen during the scattering process. In fact, more electrons are scattered in, elastically than elastically. The ratio of elastically-to-inelastically scattered electron increases with atomic number. Unfortunately, light elements constitute the major portion of biomolecules. The inelastically scattered electron creates an increased background in the diffraction pattern, especially in the small angle region. The energy lost from the inelastically scattered electron is less then 0.1 ~ of the energy of the incident beam. Therefore, the separation of these electrons from the information-carrying, elastically scattered electron is difficult. Specially designed energy filters have been used to clarify the electron diffraction patterns (ref. 8; Hui and Ottensmeyer, unpublished results). In summary, the fact that the biological membranes consist mainly of light elements, and that the differences in the mass thickness among various components of the membrane are small, do impose certain limitations in the signal-to-noise ratio of the electron diffraction patterns. These are part of the price to pay for the privilege to study the native molecules of the biomembranes instead of being confined to study the staining and replicating materials. Of course there are additional problems in specimen preservation which will be covered in a later section. In this section, we shall discuss first the applicability of the electron diffraction technique in membrane structure studies, disregarding the problem of specimen preservation.

liB. Structure analysis In relation to the incident electron beam along the z-axis, the scattering potential of a thin membrane specimen may be written as q~(x,y). The phase of the incident electron wave of wavelength 2 and energy E is modified by q~(x,y), and has the form ~p(x,y) = exp ((--i~/2E) ~ (x,y))

(3)

The alteration of the amplitude by the thin membrane specimen is assumed negligible. If the lens system is perfect, the wave in the diffraction plane can be constructed by the Huygens-Fresnel principle, to the first order [9]

•S(x*,y*) : ff(~o(x,y)) = 6 (0,0) -- (izt/2E) • (x*,y*)

(4)

where ~(x*,y*) is the Fourier transform ( J ) of the scattering potential ~(x,y), the asteriks indicating reciprocal space coordinates. If the specimen is periodic, the Fourier transform ~b(x*,y*) will have discrete values and the reciprocal coordinates may be replaced by integers (h,k). An inverse transform of ~b(h,k) gives the structure ~o(x,y) of the specimen. In practice, the electromagnetic lens in the microscope is not perfect, and the objective aperture is not infinite. The function ~P(x*,y*) must thus be modified by an aberration function, exp(iz), where Z is a function of the scattering

350 angle, the spherical aberration constant and the defocussing of the lens. Eqn. 4 then becomes

~ea(x*,y*) = ~ (0,0) - - (i:~/2E)q~(x*,y*) exp(iZ)

(5)

The first term is the unscattered beam while the complex second term gives the amplitude and the phase of the reflections in the diffraction pattern. Unfortunately, only the intensity I~ed[ 2 of the diffraction pattern can be recorded. The phase information is thereby lost. This is a common problem in diffraction with incoherent waves. Many ingenious methods have been devised to retrieve the phase information so that it can be used to reconstruct ~v(x,y). These methods, with the exception of the last one, have been widely used in X-ray diffraction and neutron diffraction [10]. They will be mentioned here only when they have been used in connection with electron diffraction of membranes and membrane-related materials. 1. The isomorphous replacement method was applied to solve the structure of copper alaninate microcrystral [11]. Attempts were made to use the lead atom to explain the structure of multilayers of stearic acid and lead stearate [12]. Thin transverse sections of multilayers of behenic acid reacted with various metal ions is a typical system where this method may be applied [13]. 2. The direct method makes use of certain statistical equalities to determine the phases of certain reflections from those of other related reflections. This method has been applied recently in the analysis of electron diffraction data of paraffins and phospholipid crystals [14]. 3. The deconvolution method is most useful in one-dimensional cases when the autocorrelation function can be deconvoluted [15]. It also applies to the case when repeat units are few (in this case the Q-function) [16]. This method has not been applied to electron diffraction analysis, but its potential in analyzing selective area diffraction is noteworthy. 4. The model refinement is particularly useful when the composition of the specimen is known. The electron diffraction of hydrated phospholipid bilayer [17] and of anhydrous crystals [18] were checked by this method. 5. The imaging technique is unique in electron diffraction since an electron image can be obtained together with an electron diffraction pattern. The phases of the diffraction reflections are obtained by comparing them with those from the Fourier transform of the image. The bright field electron image is formed by a second Huygens-Fresnel reconstruction of ~ea (x*,y*). The image is represented by [9]: ~p,m(xl,yi)= if-1 (~¢d(x*,y*))

(6)

The Fourier transform of the image intensity is:

~oa(Xl*,yl*) = ff(I~p,m(xi,yi)]2) = d (0,0) -- (2~/2E) q) (x*,y*)sinZ

(7)

The function sin Z is referred to as the lens transfer function which can be calculated theoretically from the spherical aberration constant of the lens and the amount of defocusing. The phases of ~oa(X*y*) are calculable from the image. After correcting

351 for the transfer function, the phases of ~(x*y*) are obtained [20]. This method has been used in solving the structure of catalase crystals and the purple membranes of Halobacterium halobium [19]; both of them were treated as pure phase objects. Alternatively, the phases of ~b(x*,y*) may be computed by iterating between Eqns. 5, 6 and 7 for a good match between a pair of image and diffraction pattern, as suggested by Gerchberg [21]. For irregularly packed specimens, there remains two unknowns to be solved, i.e., the unit cell structure and the disorder of the packing. The precision of one parameter must be sacrificed for the precision of the other. Since natural membranes seldomly have a perfect crystalline order, this problem is frequently encountered. The degree of disorder may be deduced from spot broadening. Broadening of electron diffraction reflections has been associated with the misorientation within each domain in phospholipid bilayers [17]. In extremely disordered cases where reflections degenerated into broad concentric bands, radial distribution analysis must be applied [22]. This is particularly relevant in the structural analysis of the lipid packing above the transition temperature. This method has been used in the study of the rhodopsin distribution in the retina disc membrane, using a negatively stained electron micrograph [23]. IIC. Limitations Most modern electron diffraction experiments - - apart from low-energy electron diffraction, which is not covered by this article - - are done on electron microscopes in order to take advantage of the existing optical setup. Therefore, the optical limitation will be discussed within the context of the electron microscope. 1. Beam coherence. Due to the finite size of the effective source and the instability of the voltage, the incident beam is not strictly spatially coherent. The diffraction pattern as well as the image ought to be modified by a function containing the coherence length, which is defined as the dimension in the specimen plane within which the incidence wave can be regarded as more or less laterally coherent. This lateral coherence depends on the divergence of the incident beam [6]. For an 80 kV beam emitted from an effective source of 10/tm in a typical microscope, the coherence length is approximately 1000 A. The chromatic coherence as limited by the instability of the high voltage is about the same order of magnitude. Information obtained from a specimen area larger than the coherent length must be corrected for the incoherence of the incident beam. 2. Selected area. One of the advantages of the electron diffraction technique is the ability to single out a small area for study. The area selection may be achieved either by restricting the illuminated area or by inserting a selection aperture in any image planes below the objective lens. The former method has the advantage of limiting radiation damage to the specimen. The smallest selected area obtainable by using a point filament and a small condenser aperture (10 #m) is approximately 500 rim. With a field emission gun, this area can be reduced further [24]. Microbeam diffraction, which is usually a feature accompanying scanning devices, may select an

352 area as small as 20 nm. However, because of the divergence of the beam in this case, the results cannot be treated as ordinary diffraction. 3. Specimen irregularity. If the extent of the regular molecular packing is smaller than the coherence length of the beam, the mosaicity of the specimen must be accounted for (see for example ref. 25). The disordering of the specimen may result from bending. For highly ordered specimens, the flatness of the specimen is very important. 4. Camera length. At conventional acceleration voltages of electron microscopes, the spacings from a fraction of an angstrom to thousands of angstroms may be resolvable with proper combinations of lens excitation. Special techniques to obtain small angle diffraction have been developed [26]. The lower limit of the diffraction resolution depends on the effective objective aperture. Were it not for the susceptibility of the specimen to the vacuum and radiation damages during the examination process, these optical restrictions would set the limits of the precision of the structural information obtainable by electron diffraction techniques. Yet, frequently, the structure of the membrane specimen cannot be preserved well enough during observation. Therefore, detailed information generally falls short of these physical limitations.

III. SPECIMEN PRESERVATION METHODS The two major difficulties in applying the technique of electron diffraction to membrane structure studies have been the requirements to subject the membrane specimens to the vacuum environment and to the electron beam irradiation. Both of these processes are known to alter the structure of the membrane, as revealed by X-ray diffraction experiments. Conventionally fixed, dehydrated, embedded and stained specimens can, of course, withstand higher doses of radiation. The negative staining technique is particularly useful in studying the planar structure of membranes. An optical diffraction of the micrographs from the stained specimen yields similar information from that obtained by electron diffraction, as discussed in the last section (Eqns. 7 and 5). Background filtering and area selection are easier to achieve in the optical diffraction method [27]. The rhodopsin distribution [23] in the plane of the photoreceptor disc, and the hexagonal phase H~ [28] ofphospholipid packing were studied by this technique [29]. However, the use of stained and dehydrated specimens has limited applications in structure analysis at the molecular level. The placement of the staining molecules may not faithfully portray the molecular organization of the membranes, not to mention the structure disruption by the dehydration process. The optical transform of the image also suffers the distortion from the lens transfer function, as previously described. Therefore, the results of the electron diffraction and the corresponding optical diffraction of dried and stained membranes are not necessarily comparable to those obtained by X-ray and neutron diffraction of the untreated specimen [30]. In order to obtain the electron diffraction

353 from innate membrane molecules, various attemps have been made to reduce the damages due to dehydration and radiation. These two aspects are discussed below.

IliA. Dehydration effects 1. Air or vacuum drying. The standard dehydration agents such as ethanol and acetone are known to extract lipids from the membranes and are thus unsuitable for molecular preservation. Many electron diffraction experiments were done on simple air-dried specimens [12,31,32]. The resulting patterns of most of these work are discrete spot patterns suggesting perfect crystalline materials. The spacing of the patterns obtained from air-dried membranes of Acholeplasma laidlawii agreed with those from its lipid extract [31] and with those from the fl-form of phospholipid microcrystals [18], indicating the crystallinity of the dried membrane specimen. Wide-angle spot patterns reported from air-dried specimens are typically those from salt crystals precipitated from the suspending solution [32]. Corresponding X-ray diffraction data on hydrated and dehydrated samples must be used to confirm the findings if it is claimed that the structure is unaltered during the dehydration processes. 2. Freezing. Apart from certain ice damage, freezing seems to preserve the fine structure of protein crystals to a degree comparable to the hydrated specimen [33]. The vapor pressure of ice is low enough for the specimen to be kept in a vacuum. In most microscopes, the specimen can be maintained frozen by the use of an anticontamination device. Keeping the specimen at low temperature has the additional advantage of reducing the radiation damage [34]. Diffraction from crystalline ice, however has to be subtracted from the diffraction pattern of the specimen. 3. Sugar substitution. This technique makes use of the amorphous sugar condensation during drying. The small molecules of glucose or sucrose may penetrate the hydrated space in a specimen. Upon 'drying', the concentrated sugar and water forms a glassy material which supposedly preserves the structure of the specimen. Sugar does not normally react with most membrane components. However, the replacement of water by an uncontrollable concentration of sugar solution after 'drying' inadvertently reduces the contrast of the specimen. The technique has been quite successful in preserving the structures of catalase crystals [19] and the purple membranes of Halobacterium halobium. Catalase crystals collapse if the hydration falls below 9 3 ~ , and diffraction patterns vanish. This method apparently can preserve the crystal structure to the same resolution as if the crystals were maintained above this humidity. 4. Environmental chamber. The environmental chamber is a device installed in an electron microscope to keep the specimen under a controllable environment. The specimen in the environmental chamber is separated from the vacuum of the microscope by a thin-film window or by a set of small (approx. 100 #m) apertures. The advantage of the environmental chamber method is that the specimen may be studied in a physiological environment. The experiment may be conducted as functions of temperature, pressure and the chemical surroundings. The major hurdle is the technical difficulties in the design and construction of the chamber. This

354 TABLE II EFFECT OF SPECIMEN PREPARATION PROCEDURE Procedure Shrinkage

Damage Ice Lipid by surface damage extractension tion

Air drying ÷

+

Freeze drying

+

Critical point drying

6-

--

Water retention

recryst- - allized

Contrast EM In situ without attach- experistain ment ment

Molecular resolution

none

?

none

?

none

?

from

dried material alone 6-

reduced none by sugar

--

__

+

reduced by ice

--

__

+

reduced environ- + by water mental stage

Sugar -embedding

--

--

(+)

Freezing

--

+

--

Environ- - mental chamber

--

--

cold stage

+

is mainly an engineering problem which can be overcome by careful designing [35,36], although in a c c o m m o d a t i n g the chamber, some loss o f resolution m a y result f r o m the alteration o f the optimal optical design o f the microscope. Phase transitions in hydrated bilayer membranes have been seen by this method [17]. D y n a m i c movements o f the m e m b r a n e [37] and the secretion process by platelets [38] have also been observed using the environmental chamber. A comparison o f these methods o f preservation is given in Table II. The absence o f the artificial contrast staining and the increased b a c k g r o u n d due to the presence o f water and its equivalence are inevitable consequences o f these preservation methods. The sacrifice is compensated for by the ability to study the innate molecular organization o f biomembranes.

IIIB. Radiation effects A t h o r o u g h discussion o f the effects o f radiation on membranes is b e y o n d the scope o f this article. However, since the beam-specimen interaction is m u c h stronger in the case o f electron irradiation than in X-ray and neutron irradiations, care must be taken to reduce any structural alteration during the experiment. Before considering the subject o f radiation damage, it is pertinent to define the term ' d a m a g e ' in the context o f structural determination. If the structural alteration by the incident beam is below the resolution o f the detection method, the damage is unmeasurable and is therefore acceptable for this method. In electron microscopy and diffraction experiments, the damage threshold is normally defined as the lowest dosage with which discernible changes in structure occur, (such as the disappearance

355 of high resolution diffraction spots), Therefore, the 'tolerable damaging dose' varies from experiment to experiment depending on the structure data of interest. Most of the radiation damages to the specimen arise from the energy transfer from the beam to the specimen, mostly in the form of ionization, radical formation and recombination from inelastic scattering events [39]. The total damage is proportional to the linear energy transfer from the beam to the specimen. From the known linear energy transfer values of common biological materials by the electron beam [40], an approximate equivalence between the beam dosage measurement in rad and the incident beam current density in A/cm 2 or in e/nm 2 can be made. The conversion factor for 100-kV electron beam incident on a thin layer of biological materials is approx. 1 rad ---- 1.6 • 10-7 e/nm 2. The conversion factor enables the vast amount of data in radiation biology to be applied to estimate the radiation effects in electron microscopy. The damage of membrane function by radiation occurs at a lower dosage than any threshold for detectable structural damages. Change in cation transport across erythrocyte membranes [41] occurs at 2 krad (3 • 10-4 e/nm2). Alterations of axonal conduction [42] and enzyme release [43] have been reported after irradiation at 5-10 krad (10 -3 e/nmZ). Molecular damage of membrane components below that detectable by electron microscopy and diffraction can also be measured by magnetic resonance [44], infrared spectroscopy [45], chemical activity [46], and electron energy loss spectroscopy [47]. The polyunsaturated lipids in membranes are very susceptible to oxidation by free radicals [48-50]. The oxidation process is accelerated when the membrane is perturbed by the so-called 'chaotropic agents' such as N O 3 - and Br-. Infrared spectroscopy studies on tripalmitin [45] show that the hydrogen elimination in CH2 occurs at a fairly low dose ( < 10 e/nm 2) whereas the breaking of the C-C bond does not occur until 60 e/nm 2. The radiation damage to protein molecules results in peptide bond breakage, enzymes with reactive - - S H groups being particularly radiation-sensitive [48]. At a dose of > 1.5 e/nm 2, catalase starts to lose its enzyme activity [46]. Amino acid composition is altered [51] by radiation as low as 10 e/nm 2. At 50-200 e/nm 2, the secondary structures of certain polypeptides (polyL-glutamate) and proteins (fl-lactoglobulin) are randomized [52]. Radiation effects become detectable by electron diffraction at a dosage of approx. 10 e/nm 2. At 50 e/nm 2, the intensities of the high resolution diffraction spots in the purple membranes of Halobacterium halobium and in catalase crystals were observed to decay [19]. Phase transitions in lipid crystal [53] were observed by diffraction at 20 e/nm 2. The low-order diffraction spots of L-valine crystals disappeared at 500 e/nm 2 under a 200 kV beam [54]. Wet, unsupported phospholipid bilayers were ruptured at a dose of 100 e/nm 2 [17]. Low temperature tends to increase the threshold, indicating the involvement of free radical formation and transport in radiation damage processes [44]. Even with an improvement factor of 4 [44] by keeping the specimen at liquid helium temperature, the threshold damaging dosage is still too low for imaging individual molecules. One must therefore resort to a high structural redundancy to obtain high resolution information. The diffraction

356 technique is most appropriate in this case since the radiation effect is spread among many unit cells. For a diffraction spot formed by a fraction F of the total irradiation, and having a threshold damage dose of N electrons per unit cell area, to stand out from the r.m.s, noise of the diffraction pattern, the number of unit ceils required should be large than I/NF 2. It should be borne in mind that the supramolecular structure in membranes, such as the collective phenomena in membrane lipids [55], phase separation [56] and the clustering of lipid molecules [57] plays an important part in the structural-functional relation. Fortunately, the interesting phenomena in membraneology can be studied with lower dosages than those required for high resolution molecular imaging. For instance, to resolve a lipid domain structure of 10 nm wide, the dosage required is only 1/100 of that required to record a micrograph having a 10 A resolution. Nevertheless, the beam current density used in the electron diffraction of native membrane molecules is still restricted to a range much lower than those used in conventional electron microscopy. It is normally achieved by using small (approx. 10/~m) condenser apertures and remote equivalent sources. There are several other techniques that help to reduce the radiation without sacrificing the information obtained. 1. The use of very sensitive detectors such as crystal scintillators or sensitive films [58,59] to improve the collection efficiency. 2. The use of an image intensification system. The present bottleneck seems to be the quantum efficiency of the phosphor screen. 3. The use of whole image electron energy filters to separate the inelastically scattered electronic background from the diffraction patterns formed primarily by elastically scattered electrons (ref. 8; Hui and Ottensmeyer, unpublished results). 4. The use of the image processing technique to suppress the background noise. This includes optical or Fourier filtering, background averaging and contrast scale manipulation by photography or by computer. 5. The restriction of the illumination area so that the portion of the specimen used for data collection is not irradiated during the optimization processes of the instrument. 6. For experiments which do not require physiological temperatures, the use of cryoprotection. This may improve the radiation tolerance of the specimen by an order of magnitude [34]. Most microscopes equipped with an anti-contamination device may be easily adapted as a cold stage for this purpose. 7. The use of radiation protective agents for membrane lipids [50] and proteins [60] to minimize the damaging effects by the irradiation.

IV. RESULTS

IVA. Electron diffraction from membrane lipids The advantages of electron diffraction in the study of the structure of a thin

357


'
Fig. 1. A. Orthorhrombic perpendicular packing of the hydrocarbon chains in phospholipid bilayers as viewed down the chain axes. B. Hexagonal packing of the hydrocarbon chains commonly encountered in phospholipid bilayers when the chains are allowed to rotate freely in the 'gel' or 'solidus' state (by courtesy of Alan Liss Inc.) [69]. layer of lipid were recognized long ago [61,62]. Paraffin and fatty acids were deposited on metallic films for diffraction experiments [61,63]. The ring spacings from these long-chain compounds were typically 4.14 and 3.67 A, which was attributed to microcrystals having an orthorhombic packing. In some cases where the crystals were large, spot patterns were seen. It was known, however, at that time, that the X-ray diffraction of paraffin crystals [64] gave spacings of 4.12 and 3.71 A. Higher resolution electron diffraction results have since been obtained from thin crystals of paraffin, fatty acids and phospholipids [18,65-68]. These paracrystals tended to be layered in nature, and in some cases resembled a multi-bilayer in structure. It is interesting to note that, as seen by electron microscopy, some phospholipids, even as anhydrous crystals, are in sheet form and are quite flexible [69]. A great deal of progress have been made on the diffraction from monolayers or bilayers of fatty acids and phospholipids, which resemble more closely the bilayer basis of biomembranes. Electron diffraction of a thin layer of membrane lipids deposited on a collodion substrate was first reported by Hurst [70]. Lipids were extracted from whole cells of baker's yeast, Escherichia coli and Staphylococcus aureus by solvents. The electron diffraction pattern showed two rings at 4.12 and 3.72 A, suggesting an orthorhombic packing of rigid hydrocarbon chains parallel to the beam (and perpendicular to the collodion support). T h i s pattern was transformed to a more diffuse 4.12 A ring after the electron beam irradiation. The author attributed this to the increasing motion of the chains from their lock-in positions in a crystal to those of rigid rotors [71,72]. This model, as shown in Fig. 1, is well known in the structural studies of long-chain compounds. Hurst also found similar patterns from phosphatidylcholine and phosphatidylethanolamine deposited on grids. Brockway and Karle [73,74] found by reflection electron diffraction that when long-chain compounds formed monolayers on a metal substrate, the chains tended to orient

358

Fig. 2. A. Electron diffraction from a hydrated and unsupported dipalmitoyl phosphatidylcholine bilayer at 30°C. The selective diffraction area is 3 #m in diameter. B. A model for the packing of the hydrocarbon chains in single phospholipid bilayers showing faults within domains and a boundary between two domains. The optical transform of the model is shown at the lower right corner (by courtesy of U.S. National Academy of Science) [17]. perpendicular to the substrate plane. Chapman and Tabor [75] observed diffraction from retracted monolayers of fatty acids. The three rings at 3.8, 4.13 and 4.5 ,~ indicated that there was para-crystalline organization within the observation area. Monolayers and bilayers of a lipid may also be formed by dipping a collodion- or Formvar-coated grid through a lipid layered on an interface. The electron diffraction of stearic acid monolayers on a collodion substrate as a function of the surface pressure of the precursor surface monolayers in a Langmuir trough was recorded by Banerjee et al. [76]. They found that the monolayer could be compressed from an amorphous structure at 15 dyne/cm to a single two-dimensional crystalline domain in the neighborhood of the collapse pressure of 42 dyne/cm. After the surface layer collapsed, the deposit picked up by the collodion film showed many unidentified rings. Similar pressure effects were observed by Hui et al. in wet bilayers of phosphotidylcholine deposited on Formvar-carbon-coated grids [77]. Electron diffraction from barium stearate monolayer deposited on nitrocellulose by the Langmuir technique was reported by Havinga and de Wael [78]. Using selective area diffraction, Glaeser and Deamer [79] obtained single crystal diffraction patterns from single domains of calcium stearate deposit on Formvar. These domains were found to extend to

359

hundreds of square micrometers. Multilayers of lead stearate were also studied by electron diffraction [12]. The patterns were fround to conform to an orthorhombic lattice of the dimensions a ---- 4.96 A and b ---- 7.38 A, as calculated from the strongest reflections of (110) at 4.13 A and (020) at 3.69 A. Electron diffraction of various behenic acid salts were recently reported by Walbillig et al. [80]. Recently, Carlemalm and Wieslander [31] reported a 'quasihexagonal' spot pattern obtained from the electron diffraction of the plasma membrane of Acholeplasma laidlawii. According to these authors, the pattern was no different from that of the lipid extract from the same membrane. The sharp spots in the pattern suggested the membrane consisted of a well-organized orthorhombic crystal, in disagreement with the X-ray diffraction of the wet specimen which suggested a hexagonal packing of lipid molecule [81 ]. Parsons observed powder ring patterns from air-dried human erythrocyte membranes and compared them to the diffraction patterns from phospholipid crystals [68]. The difference between the electron diffraction pattern of dehydrated specimens and the X-ray diffraction patterns of the corresponding hydrated specimen indicates a change of state has been induced by drying. Hui et al. [17] developed a method of forming hydrated and unsupported

360

Fig. 3. Electron diffraction patterns from hydrated and unsupported bilayers of the total lipid extracts and from human erythrocyte membrane. A. At 4.5°C, the hydrocarbon chains are in the 'liquidus' state as indicated by the diffuse ring at 4.6/~. B. At --5.5°C, some lipid has separated into a 'solidus' state as indicated by the appearance of a solid ring at 4.13 .~ beside the diffuse ring. C. A 'grainy' solid ring at 4.13/~ commonly seen from hydrolysed lipid extract and ghost membranes indicating larger patches of crystalline area. This pattern is from a hydrated ghost membrane at 2.5 °C.

bilayers of lipids on electron microscope grids. This was done by dipping fine mesh grids through the air/water interface o f a Langmuir trough under a carefully controlled environment. Using an environmental chamber, they were able to keep the bilayer stable at the temperature range o f - - 1 0 to + 5 0 ° C , by keeping it fully hydrated at all temperatures. The bilayer was covered with a thin layer of water on both hydrophilic faces, resembling the cellular environment as closely as allowed by electron microscopic observation. Using selected area diffraction and keeping the beam current below I0 e/nm per exposure, Hui et al. were able to observe electron diffraction of a single crystalline domain of a dipalmitoyl phosphatidylcholine bilayer and to measure the mosaicity and the size of the domains by varying the selective area [17]. A diffraction pattern is shown in Fig. 2A. The intensities of the (100), (110) and (200) reflections at 4.13 ~, 2.38 A and 2.08 A from the hexagonally packed hydrocarbon chains agreed with those calculated from the atomic scattering factors for carbon and hydrogen in each chain [17]. From these data a mosaic domain model for the chain packing in a hydrated phospholipid bilayer was proposed,

361

362 as shown in Fig. 2B. The crystalline spacings were measured as functions of temperature, hydration and the surface pressure of the precursor monolayer on a Langmuir trough [77]. The chains were found to orient perpendicularly to the plane of the bilayer, whereas in a multilayer, they were found to be tilted at an angle [82]. This result corresponded to earlier findings in monolayer and multilayer deposits of fatty acids on a substrate [62,74]. Thermotropic phase transition was observed by electron diffraction with the aid of an environmental chamber [5,17,36]. When the transition temperature was reached, the hexagonal patterns, as seen in Fig. 2A, were observed to transform to a diffuse ring similar to that shown in Fig. 3A. In phase separation systems such as in mixtures of phospholipids which do not co-crystallize [83] both the sharp (solid) and the diffuse (liquid) diffraction pattern may be seen to co-exist. Hui and Parsons [84] have observed this pattern in mixtures of dipalmitoyl and dilauroyl phosphatidylcholine at the phase separation temperature between 10 and 20°C. The hydration temperature-phase diagrams in general corresponded to those found by X-ray diffraction [85] and by differential thermal analysis [83,86]. In agreement with X-ray diffraction results, fully hydrated human erythrocyte ghost membranes at room temperature do not give a crystalline diffraction pattern. This also holds for the total lipid extract from the ghost membranes. The membrane lipids remain liquid-like down to the temperature of the onset of phase separation near zero degree, then a faint ring at 4.1 A appears in the diffraction pattern beside the diffuse ring at 4.6 A as shown in Fig. 3B. The evenness of the ring indicates the small crystallite size in this complex mixture of lipids at the phase separation temperature. As the phospholipids become hydrolysed, patches of larger crystallites were detected by electron diffraction. This holds also for the ghost membrane from which the lipid is extracted. With repeatedly washed ghost membranes, one can see a diffraction ring at 4.13 A derived from solid domains, as shown in Fig. 3C (Hui, in preparation). With an environmental chamber, it is now possible to follow the lipid phase transition of most membranes in a less time-consuming way. The time taken is limited only by a few minutes for the specimen to reach an equilibrium with the chamber. The onset temperature of transition of the plasma membranes of rat hepatocytes and hepatoma are found to be much lower than the physiological temperature and increasing with the deterioration of the membrane lipid [87]. The difference in the variation rates between normal and neoplastic membranes has been reported and is related to the oxidation of membrane lipids. Diffraction contrast was utilized by Hui and Parsons [84] to visualize domains of different orientations in hydrated lipid bilayers. By selectively blocking the diffraction from certain domains with a mask in the back focal plane of the objective lens, they managed to 'darken' the image of these domains. Phase-separated domains in dipalmitoyl phosphatidylcholine and dilauroyl phosphatidylcholine, as well as in dipalmitoyl phosphatidylcholine and cholesterol mixtures, were visualized at the phase separated temperature ranges. The domains of these systems (approx. 20 nm wide) were much smaller than those in the one-component systems (approx. 2/zm

363

Fig. 4. Diffraction contrast micrograph of a hydrated single bilayer of a 0.65:1 mixture of cholesterol and dipalmitoyl phosphatidyleholineat 11°C. M ~ 8000. wide). An example is shown in Fig. 4. The results and those suggested by freezefracture microscopy are comparable [56,88].

IVB. Electron diffraction from membrane proteins Many attempts have been made to obtain electron diffraction data from fibers and crystals of proteins. Electron diffraction from a film of polyamino acid fibers was first obtained by Vainshtein and Tatarinova [89]. They found a hexagonal unit cell in addition to the diffraction from the a-helix. Similar results were reported by Parsons and Martius [90] for polybenzylglutamate. The electron diffraction data agreed in general but the spacings differed slightly f r o m that obtained by X-ray diffraction, indicating that there are alterations in structure during drying processes. Parsons also obtained electron diffraction from films ofpolynucleotides [91]. Electron diffraction study of single crystals of polyglycine showed that the crystal structure was sheet-like [92]. Microcrystal plates of catalase are perhaps the most studied protein by the electron diffraction technique. High resolution data obtained by various methods of preservation have been reported [19,33,93] and detailed crystallographic analysis has been given [94]. Electron diffraction of the polysaccharide nigeran was also reported [95]. As mentioned previously, the majority of membrane proteins are irregularly arranged. If the intramembranous particles in freeze-fractured membranes represent

364

Fig. 5. Wide (top) and small (bottom) angle electron diffraction from a reconstituted purple membrane showing a hexagonal packing of protein on the plane of the membrane (by courtesy of Dr. R. Henderson and Academic Press) [19].

the true location o f certain protein molecules, the distribution of these protein is quite random. Optical diffraction o f freeze-fractured h u m a n erythrocyte membranes showed no preferred nearest neighbor distance between the intramembranous particles (Hui and Berger, unpublished results). Statistical analysis of the distribution

365

Fig. 6. The structure of a single protein unit in the purple membrane of Halobacterium halobium, based on electron diffract data from membrane discs (by courtesy of Dr. R. Henderson and McMillan Journals Ltd.) [102]. of the intramembranous particles in several membranes also revealed the true randomness in many cases, whereas in other cases the intramembranous particles tend to aggregate in irregular manners [96]. Small-angle electron diffraction of hydrated human erythrocyte ghost membranes, even with the aid of an energy filter to eliminate the inelastically scattered electrons in the small-angle region, so far failed to show any evidence of regularity of protein distribution (Hui and Ottensmeyer, unpublished results). Certain artificial systems containing high concentrations of proteins show regular patterns in electron micrographs. These systems, such as ferritin molecules absorbed in lipid monolayers [97], and cytochrome c oxidase membranes [98], are prime objects for electron diffraction experiments. The latter have been studied by optical diffraction from micrographs [99]. Unfortunately, negative staining had to be used in this case, and as a result the molecular structure was not well-preserved. However, the symmetry of the packing was clearly revealed. (See note on p. 368). The purple membrane of Halobacterium halobium is an outstanding example of

366 well-ordered protein molecules in a biological membrane, as suggested by X-ray diffraction [100] and electron microscopy [101]. The electron diffraction pattern of a large number of purple membrane pieces revealed a similarity to the ring pattern seen in X-ray diffraction; the spacings extend from 63 to 4.4/~, giving a well-ordered, twodimensional hexagonal packing. Unwin and Henderson [19] applied selected area diffraction to individual membrane pieces to obtain a single crystal diffraction pattern, which was similar to the pattern shown in Fig. 5 for a reconstituted membrane. The effects of radiation damage in this experiment was documented. The diffraction spots were phased by the image method. The transfer function was calculated theoretically as well as measured experimentally by comparing the optical diffraction of the image with the electron diffraction pattern. After correcting for the transfer function, the phases of the diffraction reflections were derived from the computed Fourier transform of a low-dose image. By using these phase information and intensities from a low-dose electron diffraction pattern, they were able to Fourier-synthesize the projected structural map of the unit cell. In addition, with the excellent data and elegant analysis, Henderson and Unwin [102] subsequently obtained the three-dimensional structure by goniometric studies. The result is reproduced in Fig. 6. The purple membrane is unique for crystallographic study due to the facts that it comprises of only one protein and the protein molecule is well-ordered in a two-dimensional crystal. Few biological membranes can match these criteria. The gap junction between cells is one of the few regular structures in cell membranes. Optical diffraction studies from micrographs of freeze-fractured and negatively stained specimens (Hui and Berger, unpublished results) as well as X-ray diffraction studies have been made [103]. Many laboratories at present are engaged in the electron diffraction studies of this membrane structure.

1VC. Electron difJraction of cell membranes Attempts to obtain electron diffraction of cell membranes have been reported intermittently during the last thirty years. Because of the difficulties of specimen preservation and radiation damage, many of these works are laden with artifacts and misinterpretations. In 1951, Fernandez-Moran [I04] reported electron diffraction patterns from osmium-fixed pieces of myelin from the sciatic and spinal root nerves of rat and frog. The patterns contained many spots of unknown spacings. The spots were much weaker in formalin-fixed specimens, indicating that the origin of these patterns were likely to be from crystalline deposits of the fixatives. Matheja [32] also found spot patterns from fixed and dehydrated myelin and retina rods of frog. The diffraction patterns did not correspond to the one-dimensional lamellar structures of these specimens and in no way resembled the X-ray diffraction results [15]. Electron diffraction of central nerve membranes from rats was reported by Khare and Mishra [105]. The pattern consisted of rings resembling those from dried crystals. The cell walls of many bacteria are more resistant to physical treatment. Taylor [106] obtained electron images from the frozen cell wall of S. serpens. If

367 an electron diffraction pattern is available, then comparison between the optical diffraction from the image and the electron diffraction can be made. Membrane stacks such as retina rod outer segments (approximately 1 ,um thick for rat specimens) are not easily penetrable by conventional voltage beams. By using a hydration chamber, Hui et al. [107] obtained diffraction patterns from single outer segments of mouse retina rods using a 800-kV beam. The diffraction spacing agreed with X-ray diffraction results [15]. With the advance of high-voltage electron microscopes, many natural and artificial membrane stacks too thick to be studied by conventional microscopes may now be studied by electron diffraction. The requirement for a coherently stacked area in electron diffraction is less stringent than for X-ray diffraction studies, due to a smaller sampling area in the former technique. IVD. Kinetic studies

To observe slow kinetics (in minutes or hours), one may quench the membrane at the desired time interval by fixation or rapid freezing. However, these methods have two serious drawbacks; the quenching procedure possibly introduces artifacts; and after quenching, sequential data cannot be obtained from the same piece of membrane. With the aid of the environmental chamber, Hui [37,108,109] was able to follow the change in diffraction patterns and the diffraction contrast images in timed sequence. At a dose level below the damage threshold for these bilayers, the change was found to be independent of the incident radiation. The rate of change was interpreted as the translation and the rotation motions of the lipid bilayer. At temperatures above the transition temperature, no sharp diffraction pattern is available to chart the motion. Labels [110] were used to aid visibility. From the two-dimensional Brownian motion, Hui [37] was able to calculate the diffusion constants of the bilayers. It was found that below the transition temperature, the translation of the molecules was mainly convective; while above the transition temperature, the motion was diffusive. The change of modes of motion corresponded to the change in diffraction patterns. The velocity of the convective motion for a bilayer of equimolar mixture of dipa|mitoyl phosphatidycholine and cholesterol was typically 3. 10-6 cm/s. The diffusion coefficient of the same bilayer above the transition temperature was found to be 10 -I° cm2/s. The results were comparable with measurements by N M R and spin-label EPR experiments [111]. Similar results on erythrocyte ghost membranes has also been reported [37]. This experiment provides electron microscopy evidence to the mobility of membrane components, as suggested by other experiments [11 I-113].

v. CONCLUSION Under certain conditions, as specified above, electron diffraction is a very powerful technique in membrane structure studies. The capability to focus the electron beam

368 gives electron diffraction the unique features of selecting a micro-observation area, and imaging the specimen. The first feature enables localized structures of the membrane to be studied, while the second feature provides phase and topographic information. The lack of these capabilities is a serious problem in X-ray and neutron diffraction studies. The main difficulties in electron diffraction are the vacuum and radiation damages to the specimen. By failing to take precautions against these aspects, many attemps to apply electron diffraction to study membrane structure have fallen into artifact and misinterpretations. The vacuum problem is a technical limitation and will be solved as technology advances. The radiation problem is a more fundamental one and poses a resolution limit to the study of unstained biological structures. The utilization of redundant information from repeat units somewhat relaxes the situation, and electron diffraction is a good example of pooling the redundant data from regularly arranged subunits. However, regular packing is an exception rather than a rule in biological structures, although identical but randomly located units within the plane of the membrane are not uncommon. An effective approach would be to utilize this type of redundancy by the method suggested by Frank [! 14]. The application of this type of technique would open up a wide area for the study of membrane structure by electron optical methods. Besides the pursuit of high-resolution molecular structure which, in most cases, is better done with X-ray crystallographic analysis of the purified component, there are many supramolecular organizations in biological membranes which are of utmost importance to the functions of these membranes. Within this category there are protein-lipid interactions, phase separation, domain and cluster structures of lipids, partition of proteins in the bilayers, mobility of membrane components, fusion, biogenesis and internalization of membranes in cells, etc. The study of these supramolecular structural-functional relations requires more careful specimen preservation and area selection rather than the ultra-high resolution capability of the electron diffraction technique. Many fruitful results may be expected to be obtained in this area in the near future. NOTE ADDED IN PROOF (Received September 14, 1977) The two-dimensional arrangement of cytochrome ¢ oxidase molecules in a collapsed membrane vesicle has recently been mapped [116].

ACKNOWLEDGEMENTS The author acknowledges the helpful discussions and comments in this manuscript from Dr. D. F. Parsons of the New York Dept. of Health at Albany, Dr. D. L. Dorset of Buffalo Medical Foundation, Drs. W. Baumeister and M. Hahn of the University of Diisseldorf, Dr. R. Henderson of Cambridge University, and Drs. H. Box, R. Parthasarathy, D. Papahadjopoulos and W. Pangborn of this Institute.

369 The c o m m u n i c a t i o n a n d permission to quote their results from Dr. H e n d e r s o n , Dr. Baumeister a n d Dr. Dorset are appreciated.

Certain results of the a u t h o r ' s

research q u o t e d in this paper were o b t a i n e d with the s u p p o r t of U S P H S grants CA15330, G M 1 6 4 5 4 a n d A m e r i c a n Cancer Society (ACS) g r a n t BC-248. Certain e q u i p m e n t used was o b t a i n e d t h r o u g h the s u p p o r t of U S P H S grant RR05648 a n d ACS g r a n t IN-54 to this Institute. The a u t h o r is a recipient of Career D e v e l o p m e n t A w a r d CA00084 from the N a t i o n a l Cancer Institute, U S D H E W .

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