Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice

Ultramicroscopy 46 (1992) 1-18 l j l ~ r , n m ~ t r r n ~ m r ~ m North-Holland

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice

Richa rd H e n d e r s o n MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Received at Editorial Office 9 April 1992

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance.

Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements . The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.

I. Introduction

It is the goal of high-resolution biological microscopy to Obtain images of individual protein and other molecules showing the internal atomic structure clearly. The realisation of such a goal would open the way for the analysis of the structure of many molecules and molecular complexes that have been intractable by other methods. In principle, it is possible to do this with the present generation of electron microscopes, except for one main problem. The electron beam, in addition to being elastically scattered by the specimen, and recombined with the unseattered beam to form an image, is inelastically scattered with consequent energy deposition in the specimen. Some of this energy deposition causes radiation damage and chemical reactions which destroy the specimen before the image has built up to an acceptable level of statistical significance. Thus, electron micrographs of proteins are noisy, be-

cause the electron dose allowed before the structure is destroyed is rather small, in the range of 1 to 10 electrons/ ,~ z depending on the temperature of the specimen.

Note that the use of heavy-metal compounds for either positive or negative staining of the biological macromolecules is generally a low-resolution method limited to determination of molecular envelopes at 20 A resolution. In high-resolution work we are dealing with unstained specimens preserved in an aqueous, hydrated and native or near-native state. It should also be noted that although electron irradiation is very damag- ing, electron microscopy is still by far the pre- ferred approach to high-resolution structure determination for small samples. The use of X-ray diffraction, for example, involves about 400-fold greater energy deposition and radiation damage per useful elastically scattered event [1]. The use of neutrons for diffraction and imaging would, in theory, be attractive because thermal neutrons

0304-3991/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved

2 R. Henderson / Image contrast in HREM of biological macromolecules

cause no damage to organic or biological specimens. However, the available neutron flux densi- ties are so low that high-resolution neutron microscopy seems unattainable for the foreseeable future.

The limiting dose allowed by radiation damage in biological electron microscopy has two main consequences. First, certain tricks which are used in conventional high-resolution electron microscopy of samples which are stable to irradiation cannot be used. Thus, normally, the specimen is exposed to a constant electron beam before any images are recorded. This allows the specimen to stabilise; any positive charge build-up due to loss of secondary electrons or other small movements of parts of the specimen due to irradiation have plenty of time to occur and the recorded image is taken with everything in a gentle equilibrium. This means that normal electron micrographs show sharp, clear pictures that

can be easily repeated. Furthermore, any loss of contrast, for example due to a poorer contrast transfer function at high resolution, can be com- pensated for by increasing the electron dose used for recording the image; the instrumental magnification can be set at any high value desired, giving a restoration of any signal-to-noise reduction due to loss of contrast. The loss of contrast, from any cause, is much more serious for work on unstained biological macromolecules, because it cannot be offset by an increased exposure; expo- sures are always limited to less than 10 elec- t rons / ,~ 2 by radiation damage.

The second consequence of limiting electron dose is that usable, low-dose images of individual protein molecules will never be possible. This is because there is not enough information in a single, low-dose image of an isolated protein molecule even to determine its position. Only if a molecular assembly of combined molecular weight

I o, inc~ent beam

Maximum specimen Proof that 1 electron dose = 5eVA 2

in di f f ract ion spot gives measurable phase in the image.

lens

Purple membrane . J strongest spot 2 x 10 .5 I O . . . . . . . . . . . . . . . . . . diffra~ion pa~ern , P ' weakest spot 2 x 10 -7 [o 1 ele

paraffin 2 x 10 -2 I 0 ~ ......

(a) ~ ~ _ (b)

n electrons

in image, F(O)- n

signal, F(g) - .Tn~T F(g) noise - ~fn

signal/noise = lO .-. phase error = 45"

Fig. 1. Schematic diagrams of the formation of diffraction pattern and image in a typical electron microscope. (a) Shows that the situation with organic and biological specimens is particularly critical because the electron dose is limited to about 5 electrons/,~ 2 for specimens at liquid nitrogen temperatures and below. The intensity of the diffraction spots, relative to the incident beam, I 0, is shown on the left. From this information, it is possible to predict the strength of the Fourier components in the image. (b) In the case where a diffraction spot, during the total exposure, would contain only 1 electron with zero background between diffraction spots, the image transform will contain the same Fourier strength, but on a noisy background which arises from the noise distribution from F(0). It is shown that the signal/noise ratio for the image and diffraction patterns are nevertheless the same,

being 1.0.

R. Henderson / Image contrast in HREM of biological macromolecules 3

greater than a few million is being studied will there be enough signal in the low-dose image to determine the position and orientation (five pa- rameters) of the molecule accurately enough to be able to average the images and so build up a noise-free average structure [2]. Hence, in smaller protein structures, there is a need for some de- gree of pre-existing order, e.g. 2D crystals, 3D crystals, or helical, icosahedral or other point group symmetry arrangement. With such an or- dered aggregate, the size of the individual particle is then sufficiently large to be able to determine position and orientation from the information contained in a low-dose image.

If, in addition, there is contrast loss for any reason, the only way to recover the structural information will be to average many independent images of different molecules so that the signal- to-noise ratio is restored to a high enough level to see the features of interest above the noise level. Thus, using 2D crystals, an atomic model of bacteriorhodopsin has been constructed [3], and 6 ,~ resolution, 3D maps of LHCII from green plants [4] and an E. coli porin, PhoE [5], have been obtained. All three of these projects required the averaging of large areas of many micrographs to restore the signal-to-noise ratio lost in imaging. In extreme cases, the degradation of the image will be such that the contrast at high resolution can never be restored with feasible quantities of data.

Thus, in the end we are always striving to improve the quality of the recorded images. Qual- ity can be defined as signal-to-noise ratio for features of interest. A particularly useful quantitative measure of achieved signal-to-noise ratio can be calculated from the ratio of the amplitude of a particular Fourier component recovered from an actual image to that which might, in principle, be obtained from a perfect image. A perfect image is one which might be calculated from scattering cross-sections assuming no degradation of image contrast from any source. In practice, electron diffraction or X-ray diffraction from samples containing large areas or large volumes of identical molecules can be used to measure the intrinsic amplitude of the Fourier components experimentally.

2. Calculation of absolute contrast

For most work involving electron microscopy of biological macromolecules, problems of dynamical scattering can be ignored and the specimen treated as a thin, pure phase object. Fig. la shows the situation for two thin organic / biological crystals: paraffin, which forms thin monolayer crystals that are very easy to make; and the purple membrane from Halobacteria, which is a real biological membrane composed of a two-dimensional array of protein molecules. In each case, the maximum dose allowed before the electron diffraction patterns fade out is about 5 electrons/~, 2, if the specimens are cooled to liquid-nitrogen temperature or below [6]. For 100 kV electrons, the intensity of the strongest diffraction spot for crystals of C36H74 paraffin is about 100 times weaker than the direct beam. For 2D crystals of bacteriorhodopsin, the purple membrane, the strongest diffraction spots are about 105 times weaker than the direct beam [7].

Fig. lb is a similar diagram which shows that if an electron diffraction pattern or an image were recorded with an exposure that resulted in 1 electron being diffracted into a particular diffraction spot, then the signal-to-noise ratio for any measurement of this spot would be 1.0. This is independent of whether the measurement is carried out in diffraction space or from Fourier analysis of an image, provided the image is perfectly recorded. It should be noted that this is a slight simplification. As shown by Henderson and Glaeser [7],

I ( g ) I g ( g ) l f(0~- - 2 ] F (0) - -~ s i n ( y ( g ) ) , (1)

where i (g ) and I(0) are the predicted amplitudes of Fourier components g and 0 calculated from the image, F(g) and F(0) are the electron diffraction amplitudes of the diffracted and un- diffracted beams and sin(y(g)) is the contrast transfer function which depends also on defocus and spherical aberration. The factor of 2 arises because, for thin untilted crystals, an electron diffraction pat tern has two Friedel-related diffraction spots F(g) and F ( - g ) of almost iden-

4 R. Henderson / Image contrast in HREM oJ" biological macromolecules

tical amplitude, which interfere with the direct beam F(0) to produce an image in which contributions from Friedel-related Fourier components are indistinguishable.

Various interesting results can be deduced from fig. 1. For example, if the weakest Fourier components from purple membrane (2 x 10 - 7 10) are to be measured from a low-dose (5 e lec t rons /A 2) image, this would require at least 1 electron in the diffraction spot. The area of crystal required is therefore (1 /5 × 2 × 10 - 7 ) = 10 (~

~2 or an area of about 1000 A square. The corresponding area to detect the strongest spot from paraffin would be only about 20 ,~2 which is slightly more than the area of one hydrocarbon chain viewed along its length. Thus, it should be just possible to see such features in an image of a single layer of 2D crystal of n-paraffin by eye, as found in the very best pictures [8-10].

When there is a loss of contrast in the image caused by any of a variety of effects, it is possible to measure the magnitude of the effect by comparing the left-hand side of eq. (1), representing the best that can ever be expected, with what is actually found in practice. If this is expressed as a ratio of image to electron diffraction amplitude, then it can be used as a quantitative index of image contrast. The remainder of this paper will present results from images of purple membrane embedded in glucose and tobacco mosaic virus (TMV) embedded in vitreous ice and discuss the findings.

3. Purple membrane in glucose

Henderson and Glaeser [7] presented the first quantitative analysis of image contrast for purple membrane and n-paraffin using the method of

analysis outlined above. The maximum contrast they obtained at 4 ,~ resolution was about 0.05. This was 5 times less than obtained from a similar analysis of images of beam-insensitive crystals of vermiculite. They concluded that there must be physical movement of the specimen during the low-dose exposure, causing a blurring of the image and consequent contrast loss. They suggested that the use of a spotscan imaging procedure using a small diameter of illumination beam coupled with a raster scan to build up an otherwise normal image might help to minimise this specimen movement induced by the beam damage. In subsequent work on paraffin and purple membrane, considerable improvements from these early results both with and without spotscan have been obtained [8,9,11-13]. Brink and Chiu [8] hold the record. Using a 400 kV microscope, with a paraffin specimen at -167°C and spotscan imaging, they achieved a relative contrast of 0.42 at 4 A resolution, which, taking into considera- tion losses due to the film, radiation damage, cryoholder, envelope functions and from electron losses through inelastic scattering, is nearly perfect. Thus it has been shown that it is possible to do well, thouogh success rates are not nearly 100%. Also, a 40 A thick crystalline monolayer of n- paraffin deposited on 100-200 A of carbon support film is likely to be a particularly stable "beam-sensitive specimen". Proceeding from this nearly idea[ situation to large virus particles embedded in thick and non-conducting films of ice without carbon support films may reveal further difficulties, as will be shown below.

Fig. 2 shows how purple membrane specimens, used for the best high-resolution images obtained so far, are prepared. Because of the need for hydrophilic surroundings, the membranes are embedded in a thin film of pure glucose, made by

embedded in l i q u i ~ _purple membrane

Carbon film

Fig. 2. Schematic cross-section showing how a specimen of purple membrane, prepared on a carbon film in glucose, may look before exposure to the electron beam. The overall thickness, including carbon film and glucose, is probably about 300 A.

R. Henderson / hnage contrast in ftREM q[ biological macromolecules 5

evaporation from the 1% solution used to wash the grids. Under these conditions, glucose is a liquid, which becomes immobile on cooling to liquid-nitrogen temperature. Fig. 3 shows a typical electron diffraction pattern and an optical diffraction pattern from an image of a similar specimen. It is clear that the contrast fades out

more rapidly in the image at high resolution. Fig. 4, however, shows that it is nevertheless possible to retrieve phases for the Fourier components of the best images of purple membrane to resolutions of 2.8 A (from Baldwin et al. [13]). Fig. 5 presents a plot of contrast versus resolution for three of the best images of purple membrane

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Fig. 3. The electron diffraction pattern and an optical diffraction panern of an untilted purple membrane specimen are shown on the same scale• It is easy to see that the image spot intensities fall off faster with resolution than those in the electron diffraction patterns. The patterns are printed with a mirror plane relating them, and with corresponding spots circled. The electron dose used was different in the two cases, with electron diffraction patterns being recorded with doses of 1 2 electrons/,~, 2 and images at

10-20 electrons//~, 2.


Fig. 4. Computer-generated plot of the intensities of the image Fourier components after extensive averaging of one of the best images (18563) of purple membrane taken in Berlin by Fritz Zemlin and Erich Beckmann. The rings show the positions of the contrast transfer function zeroes corresponding to the defocus of about 3000 ,~. The size of the symbols and the numbers, from 1 to 4, give an index of the quality of the data with the signal-to-noise ratio being 7 for a grade 1 spot and 1.75 for a grade 4 spot.

Smaller symbols are shown for weaker spots, with a symbol but no number plotted for 5 to 8.

taken over a period of several years with the best one showing contrast levels of about 50% at 10 resolution falling to 7% at 4 A resolution. Of

course, the electron dose used to record these images was high, about 20 e lec t rons/A 2 for the best image. However, despite much effort, it is


still difficult to record images of untilted purple membranes that are routinely as good as that shown in figs. 3, 4 and 5.

When 2D crystals are tilted, there are further difficulties both because of the need to prepare flat specimens and from additional contrast losses. Fig. 6 shows a comparison of contrast parallel and perpendicular to the tilt axis from an image using spotscan taken by Ken Downing of a 45 °- tilted purple membrane at - l l 0 ° C . Images of tilted specimens show much poorer contrast perpendicular to the tilt axis than parallel. Thus, even the very best image ever recorded of a tilted purple membrane (51056) still shows perpendicular less than half the contrast that it shows parallel (fig. 6). Probably only one image from thou- sands has ever shown the direction of best relative contrast perpendicular to the tilt axis, and

that specimen was tilted only to 20 ° . A good 45°-tilted specimen of purple membrane will typi- cally have 5% contrast parallel but less than 1% contrast perpendicular to the tilt axis at 4 ,~ resolution. Thus, it is much more difficult to record high-resolution images from tilted crystals, and it is the great variability that is so overwhelm- ingly striking. The effects of radiation damage cannot be directional in their degradation of the Fourier components nor are they likely to be variable, so the explanation of this problem must lie elsewhere. Obviously there is a necessity for the specimen to be flat, otherwise the spots will be blurred perpendicular to the tilt axis and the true amplitude will be underestimated - as observed. This could be the problem with bad specimens. However, where accompanying electron diffraction observations are made and the speci-

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Fig. 5. Demonstrat ion of the fall-off in image amplitudes with resolution for purple membrane. The plot shows the ratio of the image amplitude (Image amp.) to the c,t.f, corrected electron diffractkm pattern amplitude (E.D. amp). We have compared the data from the image in fig. 4 (18563) with the best film from ref. [2] (15006) as well as the best film from ref, [19] (2698). The main improvement with the better images is the slower fall-off in image amplitude with resolution giving more, stronger spots at all resolutions• Only spots with background-corrected amplitudes greater than twice the root-mean-square background are plotted. ( + ) Image 18563; (©) image 15006; ( x ) image 2698. The broken line at the top of the figure is an estimate of the theoretical level

for a perfect image.

8 R. ttenderson / hnage contrast in ttREM of biological macromolecules

mens being imaged have been shown to be uni- formly flat, there is still an enormous variation in the quality of each image, suggesting that there is another major factor at work in the case of tilted specimens. Two possibilities are substantial vertical movements of the specimen perpendicular to the supporting carbon film, and build-up of charge during the exposure, thus altering the defocus, and shifting the image in the direction perpendicular to the tilt axis. Either possibility could explain the loss of contrast and the worse effect on images of tilted specimens.

The supporting film on a typical grid square is 200 ,~ thick and 40 ~ m in span, a ratio of 2000, very similar to that of a sheet of paper. Thus it has a relatively small resistance to vertical movement even when stretched taut across a grid square. The effect of radiation-induced stresses

in the specimen could thus easily cause buckling and wrinkling of the specimen, with the vertical movement being much larger than the in-plane movement.

In the case of charging, the electron beam as it approached the top of the specimen would be attracted by the build-up of positive charge on the area being illuminated. For tilted specimens, this would result in a deflection perpendicular to the tilt axis and towards the highest part of the specimen. Similarly, the image would be de- flected in the opposite direction below the specimen. To the observer, the illumination would appear stationary, but the image would move within it, causing blurring if it were being recorded on film. Such an effect would not be a problem for normal microscope use, but only in low-dose imaging where there would be no charge at the

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Fig. 6. This shows a s imi lar plot to lha t shown in fig. 5, but fl)r an image (51[)56) t aken by Ken Downing of a spec imen of purp le m e m b r a n e t i l ted at about 45 ° to the e lec t ron beam. The data has been divided into two halves so that the contras t para l le l ( + ) and pe rpend icu l a r ( o ) to the tilt axis can be compared . The resolut ion is p lo t ted as 1/d 2 ins tead of l / d in fig. 5. This is the best t i l ted image of purp le m e m b r a n e recorded so far, and shows signif icant ly worse cont ras t in the pe rpend icu la r direct ion. Most t i l led

images are much worse than this in the d i rec t ion at r ight angles to the tilt axis.


beginning of the exposure, but a gradual build-up as the exposure proceeded.

Needless to say, it is very difficult to demonstrate which of the two possibilities is at work in any case. Of course, it is possible that both effects contribute strongly, making it more difficult to diagnose.

4. TMV in ice

A great variety of work has been done on specimens in rapidly frozen aqueous solutions and suspensions. During the last ten years, since the pioneering experiments of Dubochet, Lepault and their colleagues, many structures have been analysed to resolutions of 15-20 A. Some work at higher resolution has been done with 2D crystals in ice [14]. This shows that similar results can on some occasions be obtained for specimens in glucose and ice. In future, the use of ice as the embedding medium of choice is likely to domi- nate because it is the nearest to the native aqueous environment that is the biological norm. But how well do images of ice-embedded specimens compare to the theoretical best expected using the kind of quantitative contrast measurement outlined above?

Fig. 7 shows a schematic diagram of a specimen of TMV in ice prepared using a holey carbon grid. The TMV is suspended in a film of rapidly frozen water stretched across a hole in a carbon film. There is no conducting film of car-

bon to carry away any charge build-up. The ice, the protein and the RNA in the virus are all insulators. Any ions that were in solution initially are immobilised in the vitreous ice, and cannot move to act as current carriers to discharge any charge build-up.

Using this method of specimen preparation, many people have recorded low-dose images of TMV, and two papers have been published de- scribing the results [15,16]. Here, I will present some images of TMV taken in Cambridge, together with some quantitative analysis of these and images taken by others on a variety of microscopes.

Fig. 8 shows an image of TMV in ice. The computed diffraction pattern is shown in the inset. Fig. 9 shows a similar image of the stacked disk form of the TMV protein [17] taken by Greg Biesecker in Cambridge. An X-ray pattern of an oriented gel of TMV, kindly provided by Ken Holmes, is shown in fig. 10. The positions of seven strong diffraction maxima are indicated, one on each of the first seven layer lines. Table 1 shows the amplitudes of the peaks obtained from the X-ray diffraction pattern [18] and from a variety of images of TMV including the image shown in fig. 8. A plot of amplitude ratio of the peaks from electron images to those from X-ray diffraction is shown in fig. 11 for two typical images (that of fig. 8 and that taken from Jeng et al. [15]). There is no doubt that contrasts falls off fairly rapidly with resolution, even though there is some uncertainty in this conclusion due to the

@ii! Fiii I!iJ ii ! iIi!Kiii iii iiii!F! iii! iyii!F!iiif ii iii i!iii!iiii iii! iiiiiiiiiiii!iiiiiiii ! !iiii!i / TMV suspended within Ice film in a hole in carbon film

Fig. 7. Schematic cross-section showing how a specimen of TMV in ice is thought to be arranged. The overall thickness is difficult to estimate but probably varies from slightly larger than the thickness of the virus (200 A) up to more than 1000 ,~. The viruses are

normally photographed where they are embedded in a film of vitreous ice suspended over a hole in the supporting carbon film.

10 R. Henderson / hnage contrast m HREM of biological m acromolecules

use of X- ray a m p l i t u d e s r a t h e r t h a n t hose for

e l e c t r o n s ca t t e r i ng - bu t see next sec t ion . T h e

re la t ive d r o p b e t w e e n 25 and 12 ,~ is a f ac to r o f

5, m u c h l a rge r t han tha t o b s e r v e d for p u r p l e

m e m b r a n e images wi th s p e c i m e n s at the s a m e

t e m p e r a t u r e and a p p r o x i m a t e l y t he s a m e e lec-

t ron dose . A c o m p a r i s o n of con t r a s t fa l l -of f wi th

r e so lu t i on b e t w e e n the best image 'of T M V in ice

w i t h o u t ca rbon and the best image of p u r p l e

m e m b r a n e in g lucose wi th c a r b o n is shown in fig.

Fig. 9. An image of an aggregate of TMV-stacked disks in ice, recorded by Greg Biesecker and (inset) its computed diffraction pattern. This was recorded at 30000 × instrumental magnification and a correspondingly lower electron dose of 3 electrons/A ~. Note that there is considerable deviation from the mirror symmetry, expected to arise from the back and front projections of the particle density. Also, it is possible to see that the top end of the particle is considerably fuzzier than the bottom end. The causes of these defects are not

known at presenl.

Fig. 8. An image of TMV in ice and (inset) the computed diffraction pattern of a carefully masked off area which in- cludes a length corresponding to about half of a single virus particle (1500 ,~). This is particle 8682A in table 1, recorded at 50000x instrumental magnification and an electron dose

of 6 electrons//k 2.

12. It is c l ea r tha t con t r a s t fa l l -of f for T M V in ice

in t he best case is c o n s i d e r a b l y w o r s e than for

p u r p l e m e m b r a n e . I n d e e d (see fig. 5) t he fa l l -o f f for T M V is w o r s e t h a n tha t o b t a i n e d for p u r p l e

m e m b r a n e at r o o m t e m p e r a t u r e in 1975 [19]. Th i s

d e g r a d a t i o n in t he qua l i ty o f t he d a t a is un l ike ly

to be due to any d i f f e r e n c e in r a d i a t i o n d a m a g e .

R a d i a t i o n d a m a g e d u r i n g e l e c t r o n d i f f r ac t ion o f p r o t e i n crystals in ice is no t s igni f icant ly d i f f e r e n t

t h a n f rom the s a m e crysta ls in g lucose . In s o m e

cases, it is f o u n d tha t e l e c t r o n d i f f r ac t ion p a t t e r n s


Fig. 10. An X-ray pattern of an oriented gel of TMV, kindly provided by Ken Holmes. The original pattern shows clear layer lines out beyond 3 ,~, and the impression that the 6th layer line is considerably more intense than the 3rd is con- firmed by the numerical data shown in table 1. The first seven, strong, diffraction maxima used in the numerical com-

parisons, one on each layer line, are indicated.

3 [] J,C,S,C

• TMV-8682A

× 2

E_ • g

. ._1

0 . . . . I . . . . t , o , •

0.00 0.05 0.10 0.15 o -:L

Resolution (Angs t roms)

Fig. 11. The plot shows the ratio of the image amplitudes for TMV in ice to the X-ray diffraction amplitudes as a function of resolution. Layer line data from ref. [15] labelled J,C,S,C and from particle 8682A (shown in fig. 8 and table 1) cor-

rected for the c.t.f, are shown.

are degraded less rapidly in frozen ice-embedded specimens [14].

Many images of TMV obtained so far are similar to those shown here. They all show contrast being reduced to very low values, so that

o

resolution is realistically limited to about 10 A. B o t h t h e d i f f r a c t i o n p a t t e r n s f o r t h e p a r t i c l e s

Table 1 TMV images: layer line intensities

Layer line 1 2 3 6 Ratio 3 / 6

TMV-043 2.8 - 21.6 2.8 7.7 RM2700-3 2.1 1.6 27.4 1.4 19.6 JB2224-1 10.8 3.7 48.8 6.9 7.1 J,C,S,C (1989) 12.1 4.3 144.4 10.0 14.4 TMV-8628 1.3 0.8 18.7 2.4 7.8 TMV-8682A 4.2 2.7 32.0 5.0 6.4 TMV-EMBL-9868 - - 72.4 9.0 8.0

X-ray (JBL) 3.2 1.5 36.0 100.0 0.36

The numbers in the first four columns are the intensities of the highest peaks (see fig. 10) on four layer lines from images of TMV recorded on a variety of electron microscopes. These numbers are not corrected for c.t.f, except for J,C,S,C (1989) which are taken from Jeng et al. [15]. However, all the images analysed have defocus such that there is a zero between the 3rd and 6th layer lines (5500 ,& at 100 kV). The c.t.f, is therefore between 0.8 and 1.0 for the 4 peaks considered. Therefore, the raw numbers are a good measure of the strength of the Fourier components. The fifth column gives the ratio of the peak intensity of the 3rd to the 6th layer line. The bot tom row shows the corresponding intensities and ratio from X-ray analysis of oriented gels of TMV, from Barrington-Leigh [18].

12 R. Henderson / Image contrast in t tREM ~,j" hiolo,q, ical macromoh'cnles'

shown in figs. 8 and 9 show clear devia t ions from the helical symmetry, showing that the T M V was somehow d a m a g e d before the p ic tures were taken. This lack of precise mi r ro r symmetry shows that spec imen p repa ra t i on , even in quick-frozen, v i t reous ice, is not wi thout problems. It is not known whe the r this devia t ion from helical symmetry is due to a densi ty change dur ing f reezing [20], the exclusion of ions from the advancing i c e / w a t e r in ter face [21] or o the r unknown effects. However , my view is that at this stage, devia t ions from precise symmetry are less important to unde r s t and and should be cons ide red a s epa ra t e p rob lem from the fall-off in cont ras t with resolut ion. Note that the g rea te r s t rength of

the 6th layer line, visible in Downing ' s [9] image of T M V in glucose, nmst be largely due to con- t rast match ing of the low-resolut ion Four i e r com- ponen t s because of the approx imate ly equal density of p ro te in and glucose.

If the cont ras t loss can be overcome, then images should be expec ted to extend in resolut ion to the limit shown in the X-ray pa t te rn , which for T M V is beyond 3 A (fig. 10 and refs. [22-24]). The r e m a i n d e r of this p a p e r will discuss the possible exp lana t ions for this image deg rada t ion with the aim of p ropos ing pract ica l me thods for overcoming the p rob lem. Note that the conclusion by Jeng et al. [15], that T M V image amplitudes ag reed well with those ca lcu la ted from

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Fig. 12. Comparison of the plots of contrast versus resolution for the best TMV image (e) with similar data for the best purple membrane low temperature ( + ) and room temperature ( x ) images. It is clear that the TMV contrast falls off at considerably lower resolution, and that for this reason the purple membrane analysis has been possible to 3 ~,, whereas the TMV can only be carried

o "

out at 10 A at the present time.


atomic coordinates, is based on the coincidence of two effects they did not discuss. Namely, the calculated amplitudes at 20-30 .~ resolution were

stronger than they should have been by a factor of 3-5 because they calculated structure factors in vacuo without the inclusion of solvent, and

5

~ 4 - • • "~ . • • •

3 - • [] .E • • •

~ 2 - [] • • m • 41~ • • •

._1

0 2 4 6 8 10 12 14

= 0.5 r "

"- 0.4

i

M

E

o

r r

o .../

Resolution ~ K =)

0.3 1 1

0.2

0.1

0.0 0.0

!

,, 0.1 -t Resolution (Angstroms)

Fig. 13. (a) A comparison of the diffracted intensities from three-dimensional crystals of crotoxin, presented as the ratio of electron diffraction to X-ray intensities versus resolution. The electron diffraction data were recorded at 400 kV by Jacob Brink using a thin crystal embedded in ice (similar to that in ref. [14]), whereas the X-ray data were calculated from the precession photograph in ref. [36] taken using a much larger crystal of what is nevertheless thought to be the same crystal form. The open squares are for diffraction spots that are strong in both X-ray and electron diffraction patterns (greater than 3% of the maximum in each case), whereas the filled symbols are for spots weaker in either dataset. From the fact that the filled symbols are in general higher than the open squares, it can be seen that the distribution of intensities is such that there are relatively few weak spots in the electron diffraction pattern. This is consistent with the possibility of some dynamical scattering in the electron diffraction data since this would tend to fill in the weaker electron diffraction spots. However, it is clear that there is no large systematic change in the mean relative intensity of diffraction with resolution. (b) A similar comparison for purple membrane. Here, the electron diffraction intensities are taken from ref. [29], and the X-ray diffraction intensities are calculated from the oriented powder pattern [37], by correction for the Lorentz factor, assumed to be proportional to radius in a perfectly oriented sample. The slight increase in the ratio with resolution is probably due to the slight underest imate of the Lorentz factor due to a small amount of orientational

disorder in the specimen. Nevertheless, there is no evidence of a relative fall-off in electron diffraction intensity with resolution.

(b)

0.2

Strong spots in both

Weak spot in one


their image amplitudes were reduced approxi- mately five-fold between 23 and 11.5 A as shown in table 1 and fig. 11, and discussed above.

5. X-ray versus electron scattering cross-sections

One possible reason for a fall-off in the ratio of electron image amplitudes to X-ray diffraction amplitudes with resolution, as shown in fig. 11, could be that electron scattering amplitudes might fall off intrinsically more rapidly with resolution than X-ray scattering amplitudes. The calculated values in the International Tables for Crystallog- raphy do not take into account the effect of chemical bonding on electron scattering factors which is particularly important at the resolutions we are concerned with in biology. Also, in practice, it is difficult to record good electron diffraction patterns from TMV itself, although some results have been obtained [25].

In the resolution range being considered in this analysis, namely 0c to 8 A, diffraction arises from density differences between different structural elements.

o

At resolutions of 20-30 A, diffraction occurs due to contrast differences between protein and ice, whereas at 10 ~, resolution, diffraction occurs from, for example a-helical or /3-sheet, repeats within the structure. Many studies have shown that the contrast effects from solvent density differences have nearly faded out by 10 A resolution. Thus, in the resolution range we can so far investigate in ice-embedded specimens, we are comparing diffraction from the molecular envelope at low resolution with diffraction from the internal structure of the protein. If electron cross-sections differed relative to X-ray cross-sections when comparing ice with protein (at 20-30 A) or protein backbone with protein side chains (at 10 A) then plots such as shown in fig. 11 would be misleading.

Fig. 13 therefore presents comparisons of X-ray versus electron diffraction measurements for purple membrane and crotoxin, which are 2D and thin 3D crystals respectively. They can thus be examined both by electron diffraction and X-ray diffraction in exactly the 8-40 ,~ resolution re-

gion which is critical for the TMV in ice data. It can be seen that there is no significant trend either for increase or decrease in the e lec t ron/X- ray ratio. Certainly there is nothing of the magnitude seen in fig. 11. Note that for purple membrane, it is essentially the contrast between lipid hydrocarbon ( - C H 2 - ) and protein that gives rise to the low-resolution diffraction rather than between water and protein for crotoxin. Thus, a difference in scattering cross-section cannot contribute to the observed fall-off seen in fig. 11.

Theoretically, the mass density of water at room temperature is about 1.0 g /ml as in normal X-ray crystallography, whereas the mass density of vitreous ice at liquid-nitrogen temperature is thought to be 0.92 g /m l [26], as in the medium surrounding TMV in ice. However, there is known to be bound surface water surrounding protein molecules and in the grooves of the TMV helix. Even if all the water had altered its packing density from 1.0 to 0.92, the low-resolution amplitude increase would only be [ (1 .35- 0.92)/(1.35 - 1 . 0 ) ] = 1.23, or 1.50 on intensity, assuming a density of 1.35 g /m l for protein. The line drawn through the symbols in fig. 13a corresponds to a 1.4-fold increase in low-resolution intensity from 11.5 to 23 A resolution.

6. Discussion of possible causes of image degradation

There are at least four possible contributing factors to the poor quality of images of TMV in ice and, by extrapolation, of all other structures examined by similar methods. These are radiation damage, loss of electrons through inelastic scattering in the relatively thick ice, specimen movement and charging. Each of these factors may also be a major contributor to the remaining problems which still limit image quality for purple membrane in glucose, particularly for tilted specimens. However, since the effect is so much more severe with TMV in ice and the microscope re- quirements at 10 ,~ resolution, in terms of coherence and stability, are so much less, it should be easier to solve the problems for TMV. If this were done, then it would also be expected to shed


light on problems with other work, with specimens that are more demanding to prepare. It is unlikely that poor spatial coherence, suggested by Schr6der et al. [16] as a possible problem, plays any part in contrast loss in the 10-20 A resolution range, except at unrealistically high defocus values.

6.1. Radiation damage

Taking each of these four factors in turn, radiation damage has been the factor most thor- oughly investigated and discussed. Through study of the fading of electron diffraction spots in crystalline specimens at different temperatures, it is now known that radiation damage causes substantial structural changes in organic and biological specimens (e.g. ref. [27]). The radiation chem- istry is extremely interesting but undoubtedly complex, with different reactions occurring in different specimens. The radiation products are also likely to differ considerably at different temperatures, because even slight changes in reaction rate with temperature can alter the partition between different products. In one of the most interesting comparisons of mass loss at room temperature and at the temperatures of liquid nitrogen and liquid helium, ref. [28] showed a very much reduced rate of mass loss from collodion films at liquid-helium temperature. However, the final extent of mass loss was higher at liquid- helium than at liquid-nitrogen temperature. In spite of these mass-loss differences, the fading of electron diffraction spots has been found to be only slightly less at liquid-helium temperature [6] than at liquid-nitrogen temperature. In general, high-resolution diffraction spots fade out before low-resolution spots [19,14]. Roughly speaking, at 4 A resolution, a dose of 1 e lec t ron /A 2 at room temperature causes fading of diffraction spots from paraffin, purple membrane and crotoxin to an intensity of e - l . The corresponding dose [N e in electrons/ ,~ 2] at liquid-nitrogen temperature is about 5 e lec t rons /A 2. In one of the few comparisons between the same specimen embedded in glucose and in ice, Jeng and Chiu [14] showed almost identical values for Ne, with possibly a slightly longer lifetime for crotoxin crystals in ice.

Thus, it is unlikely that radiation damage is any more rapid in ice than in any other embedding medium. Ceska and Henderson [29] studied purple membrane embedded in sodium phospho- tungstate and found virtually the same lifetime as in glucose. It is likely that the diffusion of free radicals at temperatures of liquid nitrogen and below is over distances too small to be affected by the embedding medium. Unpublished experiments by Mark Gerstein in Cambridge, for example, showed no reduction in the effects of irradiation on purple membrane when embedded in thio-glucose, or in oxidised or reduced glu- tathione. Since thiols react with free radicals about 100 times faster than do other organic groups in proteins, the absence of a free-radical scavenging effect on electron radiation damage at liquid-nitrogen temperature seems to give further support to the idea that radiation damage in proteins will be due to internal reactions and be largely unaffected by the embedding medium. Thus, the fact that TMV is embedded in ice is unlikely to cause any greater radiation damage and is therefore unlikely to be a contributing factor in the low contrast observed at high resolution.

While it is still possible that TMV is for some special reason particularly radiation sensitive, this seems unlikely in view of the general similarity of radiation sensitivity in other organic and biological specimens.

6.2. Inelastic scattering

As the thickness of the specimen increases, the proportion of incident electrons scattered increases. Since most scattering events are inelastic (by a ratio of 3:1 for light atoms), once the ice thickness exceeds a certain value, most of the electrons will have been inelastically scattered. Also, since inelastically ° scattered electrons are delocalised by up to 20 A from the site of interaction [30] they can never contribute to the high- resolution image. For an ice thickness of 800 A, nearly half the electrons have suffered inelastic scattering [16,31]. Many of these will be delocalised, badly focused because of chromatic aber- rations in the lenses, and simply contribute to the

1(~ R. Henderson / Image contrast in HREM gf biological macromolecuh's

background fog level. In this case, they would have no effect on the resolution dependence of the data in fig. 11. However, if some of these electrons are not simply lost in a diffuse way, but contribute to the very low-resolution (20 A,) image, then they would affect such plots as fig. 11 by showing up as an apparent loss of contrast at high resolution. Not enough is yet known about the inelastic image to be able to say clearly what their contribution is. The thickness of ice in the best images is not known with certainty. The proportion of inelastically scattered electrons un- dergoing energy loss by production of secondary electrons and via plasmon scattering, and their angular distribution, is not known accurately. Fi- nally, the contribution of these different inelastic electrons to a typical image from ice-embedded TMV is not known. There are sufficient uncer- tainties here for this to require further investiga- tion. Fortunately, with the recent availability of energy-filtered transmission electron microscopes [16], some answers to these questions can be expected.

6.3. Spec imen rnot ,ement

Beam-induced specimen movement and drift are certainly the main problems in obtaining high-resolution images of untilted purple membrane and paraffin specimens [7-9,12,32]. The use of small-spot illumination and spotscan imaging has helped substantially to improve the quality and reproducibility of these images. However, we do not know whether the additional difficulties found with tilted and ice-embedded specimens arise simply from more severe manifesta- tions of the same problem or from a different problem (like charging).

In the case of tilted 2D crystals on a carbon support film, it is possible that vertical movement of the entire specimen induced by beam damage causes blurring of the image perpendicular to the tilt-axis. The dimensional similarity of a support film to a sheet of paper has already been discussed, and the much greater resistance to in- plane distortions than to out-of-plane distortions seems mechanically obvious.

Similarly, the mechanical strength of a thin

film of ice is 2-100 times less than a thin film of carbon, at least from measurement of bulk prop- erties. Therefore, it is possible that radiation damage to proteins and protein complexes in ice, together with rearrangements of the ice molecules (the products of radiation damage to water are new water molecules created from the radiolytic O H ' and H ' intermediates), simply causes greater mechanical movement, and therefore greater contrast loss due to blurring of the image. The lack of mechanical strength coupled with larger electrostatic forces arising from the lack of electrical conductivity in ice would both tend to increase any movement. Thus, specimen movement, however caused or exacerbated, remains a big problem. It is likely that the effect of physica[ movements on image quality will be ameliorated by the use of a small spotscan imaging procedure, whereas the contribution of inelastic scattering will be unaffected.

6.4. Specimen charging

Interaction of high-voltage electrons with any specimen causes secondary electrons to be ejected from the irradiated area with a consequent build- up of positive charge. After some time, the local voltage will stabilise either because ejection of further electrons is prevented by their inability to escape the local attractive positive charge, or by electrical breakdown (ionisation) and discharge through the specimen to a nearby conductor, for example, a carbon support film or a metal grid bar. However, during a low-dose exposure, the charge on the specimen will build up during precisely that part of the exposure when the specimen maintains the initial structure at its most intact; the native structure may well have decayed to a fraction of its original level by the time any charging has stabilised. It is well known that on unsupported plastic sections, imaged in the absence of an objective aperture, the specimen can charge up to the extent that it physically explodes from the internal repulsive electrostatic forces. The use of an objective aperture purely to reduce the incidence of image blurring is thus often advocated. The explanation is that secondary electrons emitted by the aperture can

R. Henderson / Image contrast in HREM of biological maeromolecuh's 17

neutralise the positive charge building up on the specimen. This is certainly very easy to demonstrate with plastic thin sections which are initially complete insulators, but does it exist as a problem with ice-embedding? Ice is not a conductor; it can be fairly thick, and it seems certain that considerable charge build-up occurs. Most micro- scopists working with ice-embedded specimens use an objective aperture for this reason. The necessity to use an objective aperture is certainly itself an indicator of the critical importance of charging. However, it is still not clear whether the residual loss of contrast with resolution is due to image blurring caused by residual charging; the image of TMV shown in fig. 8 was recorded with the use of an objective aperture. Note also that all low-dose images are recorded with the use of low-dose techniques which involve adjustment of focus and astigmatism on a nearby area distant by 1-5 /xm from the area from which the low-dose image will be recorded. Thus, during this initial focusing step, secondary electrons from the area used for focusing will be expelled and spray the surrounding area including the area to be imaged. This causes a build-up of negative charge on the area that will later be imaged. During the proper exposure, the local charge will therefore move from a negative to a positive value, making any net change in charge and its effect on the image larger.

It is claimed [33] that the application of a thin film of carbon to both sides of a thin film of ice improves the images, but this will only be proved once a real image of, for example, TMV is recorded which demonstrates superior resolution and contrast by optical diffraction or quantitative analysis. If charging is a serious problem, then it should certainly provide a clear-cut improvement. However, if it is not the most important limitation, the application of a carbon film will make the image worse, since any extra matter in the electron beam will only reduce the signal-to-noise ratio. Recently developed thin, conducting, organic films of polypyrrole could also help [34].

It is not yet clear what is the magnitude of any image contrast loss due to charge build-up on tilted 2D crystals or ice-embedded specimens. The improvements found using spotscan on tilted

2D crystals [3,9] could be caused by a reduction in either the effects of charge build-up or specimen movement.

7. Conclusion and proposal for a standard test specimen

The best images currently obtained from unstained biological macromolecules can be considerably improved. This is especially true for images of ice-embedded molecules and molecular assemblies where most of the future potential [35] of high-resolution biological structure analysis lies. Difficulties remain in disentangling the relative contributions of the various potential causes of the poor images, namely radiation damage, the contribution of inelastic electrons to the image, specimen movement and charging. In addition, the preparat ion of ice-embedded specimens re- quires further study to explain and eliminate de- viations from symmetry found with some specimens.

It is possible that the availability of a simple, standard specimen embodying many of the problems encountered with TMV in ice, but which is easily transportable and does not require cooling to liquid-nitrogen temperature, might provide a standard on which more rapid progress could be made. Such a specimen might consist of colloidal gold particles (50 ,~) or nanogold clusters (14 A) embedded in or applied to the surface of a thin film of non-conducting collodion or Formvar. Low-dose images would presumably demonstrate all the problems of radiation damage, specimen movement, charging and blurred images encountered with real specimens of biological macromolecules in ice. The presence of the gold particles would give a very much more visible image feature which could be seen at lower electron doses than at present required for the detection and evaluation of images of protein molecules or viruses. A subsequent exposure recorded follow- ing the radiation-induced conversion of the film to a conductive carbon substrate could then serve as the control experiment. Once some of the problems were worked out with this specimen at room temperature, the next step might be to


attempt a similar analysis of colloidal gold embedded in vitreous ice, finally coming back to the real problem of image contrast in high-resolution images of biological macromolecules.

Finally, of course, understanding the problem is only a beginning. Diagnosis of the cause as one or more of the above factors would then allow attention to be concentrated on possible solutions such as better specimen preparation, lower temperature stages, high-voltage microscopes with brighter and more coherent sources, or the use of better procedures to prevent or minimise buildup of positive charge on the specimen. A better understanding of the problem would allow a more rational choice of where to concentrate money and effort.

Acknowledgements

1 would like to thank several people who have generously provided the data used in some of the diagrams: Fritz Zemlin, Erich Beckmann and Ken Downing, our long-standing purple membrane collaborators, for the images from which the data in figs. 4, 5 and 6 are calculated; Greg Biesecker, whose image of TMV stacked disks is shown in fig. 9; Ken Holmes who kindly loaned the X-ray diffraction pattern of TMV shown in fig. 10; Jo Butler who supplied the TMV used to make the specimens illustrated in figs. 8 and 9; Jacob Brink and Wah Chiu, whose crotoxin electron diffraction amplitudes were used to make fig. 13a; David Scott and Paul Sigler, for lending me the crotoxin, X-ray, precession film from Achari et al. [36]. The data used to make table 1 include micrographs of TMV in ice contributed by Ron Milligan, John Berriman and Marek Cyrklaff. Fi- nally, 1 would like to thank Tony Crowther, John Berriman, Ron Milligan and Nigel Unwin for discussions and comments.

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Documents

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice