Electron Microscopy of Frozen Hydrated Biological...

JOURNAL OF ULTRASTRUCTURE RESEARCH 55, 448-456 (1976)

Electron Microscopy of Frozen Hydrated Biological Specimens

KENNETH A. TAYLOR AND ROBERT M. GLAESER

Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94 720

Received February 10, 1976

The use of frozen hydrated specimens for molecular structure determination is limited primarily by radiation damage. The radiation damage effect in frozen hydrated catalase crystals has been measured in terms of the loss of electron diffraction. The results show an improvement for this type or:specimen relative to wet hydrated or to glucose embedded catalase crystals at room temperature. Bright field images of unstained, frozen hydrated catalase crystals extend to a resolution of 11.5/~. The image resolution is presently limited by design problems with the liquid nitroge n cooled specimen stage. Rather high contrast is present in these images of unstained biological material, presumably due to the 30% difference in mass density between ice and protein. The improved resistance to radiation damage also makes it possible to give better statistical definition to the image, thereby making the image features easier to see.

The study of macromolecular structure at the level of atomic resolution is a major goal of electron microscopy. To achieve this with biological objects requires both that the structure of the specimen be pre- served unaltered in the high vacuum of the microscope and that the structural data themselves be recorded in such a manner that the effects of radiation damage are minimized. Ideally, the former condition is best satisfied by maintaining a sufficient level of hydration. The feasibil- ity of this approach has been demonstrated with both room temperature hydration stages (3) and with frozen, hydrated specimens (18). In addition to hydrated specimen techniques, it has been recently demonstrated that crystalline disorder, which usually occurs on drying, can be prevented through substitution of the aqueous phase by various sugars (21). All of these methods preserve the crystalline structure of catalase to at least 3.5 A resolution. Other methods of specimen preparation, such as embedding with water soluble compounds (12, 16), would clearly represent an improvement over conventional procedures. However, none of these methods has as yet been shown to preserve crystalline structure to the high resolution that can be obtained with wet specimens, with frozen

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

specimens, or with the glucose embedding technique.

The radiation damage problem can, in principle, be overcome by utilizing the spatial redundancy present in crystalline objects (5). This principle has been applied with great success in structural studies of the purple membrane of Halobacteriurn halobiurn (9). Although a resolution of 7 A was achieved in that study, further im- provements in electron detection and in specimen stability appear to be necessary. This paper reports on the measurement of the radiation damage effect in frozen, hydrated catalase, and on the progress of ongoing experiments aimed at obtaining image data at the level of 3.5 A resolution.

MATERIALS AND METHODS

Bovine liver catalase (C. F. Boehringer and Soehne, Mannheim) was recrystallized by two different methods. Originally, we used the procedure described by Wrigley (23), which produces thin lathes having an orthorhombic lattice. For later experiments, the method described by Dorset and Parsons (1) was used. This procedure produces thin platelets which usually have a much greater flat area than crystals produced by the former method. Both methods produce highly periodic crystals dif- fracting to greater than 3/~ resolution. Because of the greater size of the crystals obtained by the latter method, larger areas could be illuminated, which resulted in higher intensity in the diffraction patterns.

448

MICROSCOPY OF FROZEN

Two methods have been successfully used for pre- par ing frozen hydrated specimens. "Sandwiched specimens" are prepared on 200-mesh folding grids covered with a support film of carbon, with about 10 to 15 A of SiO vacuum deposited onto the "specimen side" of the carbon film (17). Silicon monoxide is used to impar t a hydrophilic character to the other- wise hydrophobic carbon film. Although two stand- ard grids can be used, folding grids are preferred for the purpose of al igning the grid bars. The procedure used is shown in Fig. 1. The 200-mesh grid is par- tially folded (Fig. la), and a suspension of catalase crystals is placed between the two halves (Fig. lb). The grid is then folded completely, the excess water wi thdrawn with filter paper (Fig. lc), and the specimen frozen immediately in liquid nitrogen. The specimen is manipulated into the specimen holder under liquid nitrogen, using specially constructed forceps.

The second method of preparat ion produces frozen specimens tha t are mounted on single support films instead of being sandwiched between two sup-

/i "~\

w / \ / \ k £ \

! \ / \

FOLDING ELECTRON MICROSCOPE GRID

HYDRATED SPECIMENS 4 4 9

port films. Again, 200-mesh grids with SiO-coated, carbon support films are used. The grid with a drop of catalase suspension on it is held by a forceps inside a minia ture humidi ty chamber, which is mounted on the stage of a phase contrast l ight :microscope. Humidified air flows continuously through the chamber. As much excess water as possible is removed from the specimen with filter paper while i t is held in the chamber, and the remainder of the water is then allowed to evaporate slowly. Even- tual ly a point is reached where the water film be- comes so th in t ha t Brownian motion ceases. With fur ther evaporation, thickness fringes can be observed in the remain ing concave water film. When these fringes are observed in a majority of the win- dows, the specimen is removed from the chamber and frozen directly in liquid nitrogen.

Prefrozen specimens were introduced into the vacuum of the JEM 100B electron microscope by using a combination cold sink, frost protector which mounts in a modified airlock door (19). A commer- cially bui l t JEOL liquid nitrogen-cooled stage with

b.

MICROPIPETTE

C.

EXCESS FLUID DRAWN OFF WITH FILTER PAPER

d,

GRID PLUNGED INTO LIQUID NITROGEN

LIQUID NITROGEN BATH CONTAINS SPECIMEN HOLDER CAP

X~L 7410-8109

FIG. 1. Procedure for producing-frozen th in specimens for electron microscopy. (a) A 200-mesh folding grid is part ial ly closed. (b) A drop of a suspension of specimen sufficient to fill the space between the two faces of the grid is applied. (c) The two faces of the grid are folded tightly, and the excess fluid is wi thdrawn with filter paper. (d) The specimen is immediately frozen by plunging into liquid nitrogen, and the grid is placed into the specimen holder cap. The cap is mounted upr ight in a brass block, which is also submerged in liquid nitrogen. The cap with the specimen is mounted onto the specimen holder, which is then placed into the cold sink-frost protector for t ransfer into the microscope.

450 TAYLOR AND GLAESER

an attainable specimen temperature of -120°C was used. A 20-t~m condenser aperture was used to obtain beam intensities of about 10 -5 A/cm ~ at 100 keV.

Beam current densities were measured with a lithium drifted silicon detector mounted just below the final image plane of the electron microscope. The efficiency of this detector for 100 keV electrons has been estimated to be 80% (10). However, the data reported here have not been corrected for detec~ tor efficiency. Current densities were measured through gaps in the support film, and they have been corrected for the final image magnification at the detector plane.

Electron diffraction patterns were recorded on Kodak Electron Image Plates and, in some cases, on Kodak No-Screen X-Ray Film. Electron image plates were developed according to the manufac- turer's instructions for maximizing the electron speed. The X-ray film was developed as described by Matricardi et al. (14).

Imaging experiments were carried out in the fol- lowing manner. Specimen grids were scanned with a spread beam at low magnification (5000×) until a crystal was located in an appropriately thin envi- ronment of ice. Because of the size of the crystals, it was usually possible to examine one part of the crystal and yet record the image from a previously unexposed area. Ice extending over the crystal could be identified by virtue of the bend contours present. Catalase crystals that were surrounded on their edges by ice, but which did not appear to have ice extending over the surfaces, were the most favora- ble candidates for lattice imaging. Minimal exposure (22) images were recorded at magnifications of 30 000x on electron image plates, using plate exposures that usually were of the order of 0.1 electrons/ /~m 2.

RESULTS

Al though radiat ion damage can be measured by a var ie ty of techniques, for crystalline objects the effects can be deter-

m i n e d most easily by recording changes tha t occur in the electron diffraction pattern. These changes are usual ly mani- fested by a gradual loss in the resolution to which crystall ine diffraction extends and by a reduction in diffraction intensi ty with increasing electron irradiation. The measurements reported here a r e concerned pr imar i ly with the total electron exposure tha t causes the complete loss in crystalline diffraction at a given resolution; this exposure is referred to here as the ~critical exposure" for the specified resolution.

Figure 2 i l lustrates the changes tha t oc-

cur in electron diffraction pa t te rns of frozen hydra ted catalase crystals as a result of radia t ion damage. The diffraction pat- te rn shown in Fig. 2a extends to 2.8 /~ resolution, which is indicative of the h igh degree of order tha t can be obtained in these crystals. After the same crystal had received an exposure of 2.5 electrons/A 2, the resolution to which the pat tern extends was reduced to about 3.6 fl~. In addition, there is an overall reduction in diffraction intensity. Fu r the r reductions in the diffraction intensi ty and in the resolution are evident after exposures of 5.0 and 11 electrons//~ 2. I t is significant to note tha t even after an exposure of 11 electrons/ /~2, the diffraction pa t te rn still extends to 8.5 A resolution.

Data from several exposure series similar to the one shown in Fig. 2 are combined in Fig. 3, which shows the crystal resolution remain ing in the electron diffraction pa t te rn as a function of the accumulated electron exposure. Included in Fig. 3 are data obtained using both types of crystalli- zation procedures and both types of frozen specimen preparat ion procedures. The scatter in the da ta points shown in Fig. 3 is due pr imari ly to individual differences between crystals, ei ther in crystal dimen- sions or in crystal quali ty prior to irradiation. As previously mentioned, the larger crystals obtained, using the procedure of Dorset and Parsons (1) , permit one to obtain more intense diffraction patterns. As a consequence of the h igher initial intensity, a greater total exposure is necessary to reduce the diffraction to an immeasur- able level. We have noticed tha t diffraction pa t te rns tha t did not extend to high resolution initially were relat ively insen- sitive with respect to loss of resolution until h igh exposures were accumulated. We could observe no significant difference in radiat ion sensit ivity between sandwiched and ~'open face" specimens.

Figure 3 shows a s t ra ight line which is the least-squares best fit to the experimental data. F rom this line the accumulated electron exposure tha t results in complete

MICROSCOPY OF FROZEN HYDRATED SPECIMENS 451

FIG. 2. Changes in the electron diffraction pat tern of frozen, hydrated catalase crystals resul t ing from radiation damage. (a) The ini t ial diffraction pa t te rn extends to 2.8/~. The r ing present in the pa t tern at a spacing of 3.67 A is due to ice condensed on the surface of the specimen from water vapor present in the column of the microscope. (b) The pat tern recorded after an exposure of 2.5 electrons/A ~. (c) The pat tern recorded after an exposure of 5.0 electrons//~ 2. (d) The pat tern recorded after an exposure of 11 electrons/A~; the diffraction pat tern still extends to 8.5 A resolution.

loss in crystalline diffraction is 27 _+ 9 electrons/A 2. Compared with the value at the same end point reported for hydrated catalase at room temperature, namely, 2 electrons/A 2 (13), this represents about a 10-fold improvement for the frozen catalase crystals. The published data on the loss of intensity of the (0, 4) reflection in glucose embedded catalase specimens as a function of electron exposure suggest that

an accumulated exposure of 4 electrons/A 2 will result in complete loss of diffraction by this type of specimen. Our own measurements on this type of specimen have also resulted in the value of 4 electrons/A 2 as the critical exposure for complete loss of the diffraction pattern at room temperature. At -120°C, the critical exposure for glucose embedded catalase improves to a value of about 12 electrons/A 2.


0 . 5 3 . 5

m O J I 0

- - ~ I I ] I I ] 1 I I I ] I [ 1 1 I O, t 1 .0 10

Accumulated exposure ( e - / ~ 2)

Fie. 3. Crit ical exposure for fading of the electron diffraction pattern of frozen hydrated catalase as a function of the resolution.

Curiously, the electron exposure that will reduce the resolution of the diffraction pattern in the frozen, hydrated catalase crystals at 15/~ resolution compares favor- ably with the reported value for uranyl acetate-stained catalase [12 electrons//~ 2 (5) ]. Any comparison between the two types of specimens ends at this point, because the negatively stained specimen reaches a final, stable end point beyond which no further changes in the resolution of the diffraction pattern occur. With the frozen catalase crystals, this type of limi- tation to the damage does not occur. Very gross radiation damage effects can be observed when specimens are irradiated with very high beam intensities (17).

Our radiation damage measurements suggested that bright field imaging of frozen hydrated specimens could be easily ac- complished. The type of image that can be obtained at the present time is demonstrated in Fig. 4, which is a lattice image of an unstained, frozen hydrated catalase crystal. In the present state of development, we are able to obtain image resolutions comparable to what is obtained in negatively stained specimens. The contrast is quite high for an unstained specimen, although it is not nearly as great as for a negatively stained specimen. Certain periodic features are visible in Fig. 4. This demonstrates that at least some spatial

frequencies have been imaged with statistical definition, as defined by the Rose equation (5). For most of the spatial frequencies shown in the optical diffraction pattern of this image, the information is spread over many unit cells in a statistically noisy fashion. Thus, if any conclu- sions about the structure of catalase were to be drawn from Fig. 4, spatial averaging would be necessary. This requirement, which is likely to hold for any unstained specimen, apparently is also needed for many negatively stained specimens since it has been shown that high resolution images of negatively stained catalase crystals can also be statistically noisy (11).

DISCUSSION

The observation that a high degree of periodicity can be maintained upon freezing a crystalline protein such as catalase is somewhat surprising, considering the many reports of ultrastructural damage that can occur when biological material is frozen. The damaging step in the freezing of a protein crystal is believed to be due to the formation of crystalline ice within the protein lattice (20). Because the density of crystalline ice is less than that of water,

t h e resulting expansion during freezing causes loss of three-dimensional order of the protein. It should be pointed out that in the experimental system that we use, the formation of crystalline ice can occur either during the initial freezing step or in the subsequent warming of the specimen to the cold-stage temperature of - 120°C.

To prevent crystalline ice from disrupt- ing a protein lattice during freezing, several methods have been successfully employed in the past. Among these are the use of high concentrations of sucrose (6) or glycerol (7) to act as cryoprotectant and freezing under high pressure (20). Freez- ing at high pressure permits ice to crystal- lize, but in a high density phase. In our case, success is apparently achieved because the cooling rate is fast enough to freeze the water into a vitreous (glassy)


FIG. 4. Bright field image of an unsta ined frozen hydrated catalase crystal. The optical diffraction pat tern in the insert indicates the presence of the (6,0) reflection at 11.5/~ resolution.

state. The freezing rate for a very thin water film in liquid nitrogen is unknown, but in at least one report (8) it is shown to be sufficiently fast to indeed attain a vitreous state. Vitreous ice films, if formed, will not be observed in the electron microscope unless the temperature of the cold stage lies below the temperature for the vitreous-to-crystalline ice-phase transition, about -130°C (3). This condition is not satisfied in our case.

Whether 'the bulk aqueous film sur- rounding individual protein crystals freezes in a vitreous state or not, the formation of crystalline ice within the aqueous channels of the protein l a t t i ce - either during freezing or by subsequent w a r m i n g - c a n be prevented by virtue of

the ~cryoprotection" that the surface of the protein molecule provides. The ability of a polypeptide to inhibit ice formation is well documented in the X-ray diffraction work of Dowell et al. (2) with gelatin gels. Their results show that with increasing gelatin concentrations and increasing cooling rates, there is a trend toward the produc- tion of vitreous ice at the expense of the various crystalline phases. When the cold gels are warmed, the temperature at which the transition from vitreous to crystalline ice occurs increases with increasing gelatin concentration from about -55°C for a 50% gel to -45°C for a 58% gel. At 60% gelatin and above, no crystalline ice is formed, either during freezing or rewarm- ing with any of the rates used. These re-


sults support our belief that (1) even if the freezing rate is not fast enough to freeze the thin aqueaus films into vitreous ice, it is fast enough to freeze the aqueous channels within the protein lattice into a vitreous state, and (2) if the aqueous films initially freeze into a vitreous state and then recrystallize on warming to -120°C, the cryoprotection afforded by the polypeptide chain prevents this from occurring within the catalase crystal lattice.

The comparison of a protein crystal and a gelatin gel is limited somewhat by the structural difference between these two forms of polypeptide. The folded, compact nature of the globular protein, as opposed to the extended, open structure of the gel, will result in a reduced efficiency as a cryoprotectant per unit weight for the catalase molecule. This reduced efficiency may be adequately compensated by the vastly increased freezing rate that is achieved with our specimens compared with the fastest rate employed by Dowell et al. (2), and by the temperature of our cold stage which lies comfortably below the temperatures at which they observed the transition of glassy to crystalline ice.

The specimens that we observe in the electron microscope are clearly not lyophi- lized material. Abundant evidence exists to support this assertion. First of all, the catalase crystals are normally in contact with a thin, crystalline film of ice, which is stable for an extended period of observation except at very high beam intensities. Secondly, condensation rather than subli- mation occurs, resulting in the buildup of microcrystals of what is believed to be cu- bic ice (see Fig. 2).

The increased resistance to the effects of radiation damage that we observe in frozen hydrated catalase crystals is much larger than has previously been observed in low-temperature studies of small molecule organic specimens (15) and is proba- bly due to the mechanical supporting ac- tion that the ice matrix provides. The ra-

diation damage process that occurs in the electron microscope is believed to take place in two stages (15). The primary event (believed to be temperature-inde- pendent) is bond fracture, which results in the formation of various radical species. The secondary step is molecular rearrangement or diffusion of the fragments resulting from bond fracture. This second step is temperature-dependent and will occur quite easily for a protein molecule in a liquid matrix. By converting these aqueous channels in the crystal to a solid, this diffusion or rearrangement process will be reduced substantially, and the protein conformation might be maintained up to a higher dose of irradiation.

The improvement with respect to radiation damage that we observe in electron diffraction patterns of frozen catalase crystals is similar in magnitude to one reported for X-ray diffraction patterns of sucrose embedded lactate dehydrogenase crystals at -75°C (6). In addition, we observe threefold enhancement in crystal lifetime with glucose embedded catalase crystals at -120°C. This improvement can be explained if it is assumed that the glucose-filled aqueous channels are fluid rather than solid at room temperature, a suggestion that has previously been made by Unwin and Henderson (21). If the enhancement in crystal lifetime is indeed due to the conversion of the fluid aqueous channels to the solid state, then we would predict only a small improvement in the critical exposure if the specimen temperature were lowered still further to liquid helium temperature. This prediction sup- poses that the hydrated protein crystals will behave in a way similar to what has been found for other organic crystals once the fluid in the protein crystal has been solidified by freezing.

All the lattice images of unstained, hydrated catalase crystals that we have obtained have surprisingly high contrast at low resolution. This high contrast arises primarily due to the difference in mass


density between protein and the surround- ing aqueous p h a s e - t h e densities of hydrated proteins normally being about 33% greater than that of water. This mass density contrast will only be visible at lower resolutions where the protein lattice has large, well-defined aqueous c h a n n e l s - a situation that is generally observed in protein crystals. In hydrated catalase crystals, the small angle diffraction pattern is dominated by the (0,2), (1,1), and (2,1) spots which must arise from large regions within the lattice having greatly different scattering potentials.

Our experience with hydrated catalase crystals is somewhat different from that rePorted for glucose embedded crystals. In the latter case, the aqueous channels are filled with glucose medium of an unspeci- fied density, certainly greater than 1.0g/ cm 3. This essentially reduces any contrast that may have been due to a density difference between the protein and the aqueous channels. It should also be pointed out that the lattice images described here are usually recorded with a somewhat higher specimen dose than that utilized by Unwin and Henderson (21), and this higher electron exposure further contributes to the image definition.

The contrast present in images of frozen hydrated catalase crystals is a definite advantage for low resolution imaging. At higher resolutions, however, image contrast arises from fluctuations in density within the protein material itself. Thus, from the standpoint of contrast, there will be no advantages of this method of specimen preservation for higher resolution imaging. However, from the radiation-damage standpoint, the frozen hydrated specimens will have a small relative advantage for high resolution imaging. At the present time this potential advantage is unfor- tunately offset by the apparent lack of a cold stage that has sufficient mechanical stability to provide high resolution micro- graphs.

The mechanical instability present in

our liquid nitrogen-cooled stage can be shown to be one of vertical vibration, which effectively causes the focus to rap- idly vary. This effect can be demonstrated with optical diffraction patterns from images of thin carbon films recorded at low temperature. Such optical diffraction patterns are invariably circularly symmetric (in the absence of drift and astigmatism). If lateral vibration were present, the optical diffraction patterns would be noticea- bly asymmetric (4).

The technical problems of stage stability may be difficult to overcome at 100 keV, but might be solved more easily by using higher voltages since the greater depth of focus at high voltages will substantially reduce the tolerance that must be met in order to still obtain high resolution images.

The authors wish to thank Mrs. Grace Walpole for the preparation of the manuscript.

This research was supported by Biomedical Sci- ences Support Grant NIH-505-RR 7006-10, USPHS Training Grant 5T01 GM 00829, and by the U.S. Energy Research and Development Administration.

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