Scanning tunneling and scanning transmission electron microscopy of biological membranes

Surface

394

Science 181 (19X7) 394-402 North-Holland, Amsterdam

SCANNING TUNNELING AND SCANNING TRANSMISSION ELECTRON MICROSCOPY OF BIOLOGICAL MEMBRANES A. STEMMER,

R. REICHELT,

A. ENGEL

M.E Miiller-Institute

for High-resolution Electron Mwoscop~~ at the Blozentrum of the Univem~v of Basel, Klmgelhergstrusse 70, CH-4056 Basel, Switzerland

J.P. ROSENBUSCH Department of Microbiology, CFI-4056 Basei, Switzerland

M. RINGGER, Ph_vs,srcsInstitute Received

Biozentrum

H.R. HIDBER

of the Unwerslty

of Bosei, Klingelhergstrusse

and H.-J. GUNTHERODT

of the Unruersrty of Base{, Klingelhergstrusse

14 July 1986; accepted

70,

for publication

30 October

82, CH-4056

Bcrsel, Swrtzerlmd

1986

The feasibility of imaging porin membrane, which is a reconstituted biological membrane consisting of phospholipid and protein, was studied by scanning tunneling microscopy (STM). Due to detailed knowledge of its composition from biochemical and its three-dimensional (3D) structure from electron microscopical analysis, porin vesicles seem to be a suitable model specimen for exploring the application of STM in biology. Unstained vesicles adsorbed onto a thin amorphous carbon film supported by a finder grid were localized using a scanning transmission electron microscope (STEM) at low irradiation doses ( < 100 e -/nm’). Suitable areas of the sample were then positioned in the STM by a light optical telescope. STM images taken under ambient pressure from empty amorphous carbon films exhibited corrugations in the range of 5 I nm, whereas steps having a height of 5 nm were reproducibly observed on grids with porin vesicles. Since this value is in good agreement with that obtained from air-dried metal shadowed vesicles, we interpret these steps as the edges of porin membranes.

1. Introduction Micrographs of single atomic steps recorded by the scanning tunneling microscope (STM) [l] shortly after its invention and STM images of unprecedented clarity revealing the atomic structure of solid surfaces (for reviews see, e.g., refs. [2,3]) are rated as some of the most exciting of last decade’s experimental results in physics. More recently, the possibilities of this new kind of microscopy have been explored for the analysis of biological macromolecules and their supramolecular assemblies, in spite of the fact that biological structures lack two properties which could be essential for obtaining 0039-6028/X7/$03.50 0 Elsevier Science Publishers (North-Holland Physics Publishing Division)

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clearly interpretable atomic resolution micrographs of their surfaces: neither are they sufficiently flat nor do they conduct electric current. Nevertheless, initial images of DNA [4] or bacteriophage $29 structures [5] adsorbed onto graphite and air-dried demonstrate that “topographs” from such objects can be obtained by STM. Encouraged by these results, we started to analyse membranes reconstituted from E. coli outer membrane porin and phospholipid [6] by STM, whose three-dimensional (3D) structure we have determined recently to a resolution of 2 nm by electron microscopy (EM) and digital image processing [7]. With the goal to investigate whether a correlation between structural data obtained by EM and the STM topography exists, we air-dried unstained porin membranes adsorbed onto thin carbon films. Dark-field micrographs recorded at doses well below 100 e-/nm’ using the scanning transmission electron microscope (STEM, [S]) then allowed well preserved, flattened membrane vesicles to be identified and to subsequently be positioned under the scanning tip of the STM. 2. Materials and methods Porin membranes were reconstituted as described previously [6] and adsorbed onto a thin carbon film glow discharged in air at low pressure. The carbon film was supported by a thick, fenestrated carbon film that was mounted on a finder grid (Maxtaform H-2). Samples were thoroughly washed in quartz-distilled water, air-dried and either transferred directly into the STEM or platinum/carbon shadowed at 30° for investigation by conventional transmission electron microscopy (CTEM). Suitable membranes were localized in the STEM at a magnification of < 20000 X and a dose of < 100 e-/nm’. As our STEM currently does not provide the possibility for in situ STM measurements, the finder grids had to be transferred to the STM, where areas of interest were repositioned using an optical telescope and a 2D electromagnetically driven “walker” [9]. The finder grids with a mesh size of 120 pm were indispensable for the alignment procedure resulting in a precision of approximately 10 pm. From this precision and the average size of porin vesicle clusters the probability to find a membrane with the STM was estimated to be at least 100 times better than without positioning the sample. STM images of approximately 100 x 100 nm’ were digitally recorded for further processing and displayed by an x-y recorder at the same time. 3. Results and discussion Fig. 1 shows air-dried porin membranes after unidirectional shadowing with platinum/carbon, revealing edges of and a random texture within the vesicles.

Fig. 1. Unidirectionally platinum/carbon shadowed (30’) air-dried porin membranes exhibiting single and multi layers. The polystyrene sphere is used to calibrate the magnification and to determine the shadowing direction. Bar represents 100 nm.

After freeze-drying and shadowing a regular surface relief can be observed [7] suggesting that air-drying introduces severe collapse due to surface tension [lo]. From shadow length and direction calibrated via the shadows casted by polystyrene spheres of known size, the step height of air-dried vesicle edges is calculated to be either 3.8 f 0.6 nm (94 measurements) for single steps and, depending on the type of edge line measured, 7.1 + 0.7 nm or 10.2 f 0.8 nm (112 measurements) for double layers. Single membrane layers are not thinner than 3.9 nm which is the thickness of the lipid double layer, but after shadowing their height is rather underestimated due to the size of platinum/carbon clusters. In the case of double layers, the larger of the two step heights might be due to a slight displacement of the upper and lower

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Fig. 2. STM image of clean amorphous carbon film prepared by evaporating spectroscopic grade carbon on freshly cleaved mica. The image was recorded with U,, = 0.8 V and l,i, = 1 nA at ambient pressure. Corrugations amount to 0.5-l nm.

membrane as observed by STM (fig. 5 below) which results in longer shadows, Fig. 2 illustrates a STM image of the thin carbon film used as substrate of porin membranes. In agreement with published data [ll] its surface roughness exhibits a smooth, random nature with an amplitude of 0.5-l nm, sufficiently small for STM studies of biological samples. Such images were easily and reproducibly obtained. The sequence of micrographs shown in fig. 3 illustrates how unstained pot-in vesicles are localized on the finder grid by dark-field STEM. STM images recorded from that area reveal distinct steps besides the typical carbon film structure. Although such steps were frequently measured, the fields recorded were too small to identify whole membrane patches. In one case however, two distinct, slightly curved steps can be discerned (fig. 4). The tip voltage was varied during the scan over a range of OS-l.4 V in order to

,;ig. 3 Light optical image of a copper finder grid (Maxtaform H-2) which has a mesh size of approximately 120 pm (a). Annular dark-field micrographs digitally recorded with the STEM at X0 keV. diffcrcnt magnifications and electron doses below 100 e /nm’ (b)-(d). Membrane vesicles marked by A and B, respectively. can easily be identified by their constant image intensity within the vesicle and their roundiah shape. Thick areas like the holey carbon film yield a very high image intensity which gives an overflow of the digital counting electronics, thereby generating a black/white pattern. The frame in (b) marks the area shown in (c).

study its effect on the typical image features, which were found to be independent of tip voltage (fig. 4a). While the empty carbon film looks similar to the one shown in fig. 2, steps of the kind displayed in fig. 4 could only be observed on finder grids containing porin vesicles. The height of the first step in fig. 4 estimated from 27 line scans is 5.0 + 0.6 nm; similar edge lines traced at a tip voltage of 1.8 V

A. Slemmer et al. / STM and STEM of biological membranes

Fig 4. (a) film taken voltage of z-direction.

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STM micrograph of the edges of a porin vesicle adsorbed onto an amorphous carbon at tip voltages from 0.5 to 1.4 V and a tunneling current of 1 nA. Areas of constant a continuous scan are shown as individual blocks with an arbitrary shift in the (b) The frame of (a) is converted into half-tones such that high intensity corresponds to high elevation.

(data not shown here) yielded step heights of 5.0 + 0.5 nm and 10.7 + 0.3 nm (12 and 6 measurements, respectively). These data correlate well with the result from shadowed air-dried vesicles (fig. 1). A second, somewhat smoother step runs parallel to the first in fig. 4 at a distance of 15 to 20 nm. We interpret this as the edge of a flattened porin vesicle with the lower and upper membrane disrupted and slightly displaced during air-drying. Their shape is confirmed by STEM images, where such roundish edges are frequently observed (fig. 3). The influence of the work function is estimated from the relation I -

(U/s)

exp( -A+11/2s),

where I is the tunnel current, U the tip voltage, A a constant, @ the barrier height, and s the distance between tip and sample. Considering the work functions of carbon and a lipid bilayer (4.6 and 5.1 eV [12], respectively) it becomes evident that the apparent step height of 5.0 nm is only insignificantly modified by the change of the work function. In addition we would expect the difference of the work functions to be further decreased due to adsorption

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layers. Taking into account the reproducibility of carbon film images, the frequent occurrence of steps when porin vesicles are present, and the good correlation of step heights measured by STM with those from shadowed samples, the surface relief shown in fig. 4 is most likely the edge of a membrane contoured by the tip of our STM. Even if STM micrographs reflect predominantly topographic features of biological structures, their interpretation appears less straightforward than for inorganic crystals. Besides preparation artifacts during air-drying that are known to change the surface relief of biomacromolecules in an unpredictable way [lo], and the sparse knowledge of their electronic properties, the scanning tip itself may generate artificial features related to its particular shape. Generally, atomic resolution is achieved with a tip radius of 0.5-l nm; ideally the tip consists of a single atom only. These tip characteristics are sufficient for STM studies of crystal surfaces with topographic elevations -C 2 nm. However, biological structures like single molecules (3-20 nm), oligomers (lo-30 nm), membranes (4-5 nm thick), and viruses (20-100 nm in diameter) exhibit surfaces which are significantly less flat. From fig. 5 it becomes clear that the geometry of a typical tip prepared by grinding must have a significant influence on the STM image of large biological structures. It should be noted,

Fig. 5. CTEM micrographs of a mechanically ground tungsten tip after tunneling. The tip where tunneling took place is marked by an arrow (a), (b). The mean tip radius amounts to - 35 nm (c). On top of the tunneling tip there are two “nanotips” spaced by X nm.

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however, that from such and other tips studied in the CTEM, near atomic resolution STM images of crystalline or amorphous solids were reproducibly obtained. The finger-like protrusion of the tip shown in fig. 5 represents the part where tunneling was likely to occur. Its mean radius is 35 nm, but on its top two “nanotips” with radii of < 1 nm can be discerned. Their separation related to the tip axis is 8 nm. On atomically flat surfaces only the outermost of the “nanotips” acts as tunneling tip, whereas on a rough surface such as porin membrane, both of them may collect tunneling electrons, depending on the local topography. For thicker structures such as bacteriophages the situation becomes even worse. In case of a T4 phage with a vertical extension of 70-80 nm, other protrusions may start to tunnel before a “true” image can be recorded by the outermost tip. Similar tip geometry effects were observed and discussed in refs. [13,14]. It therefore seems highly desirable to know the geometry of the tip as accurately as possible when studying biological samples. We have used porin membranes as test object to investigate the possibilities of STM for probing the surface topography of biological samples. Steps of 5.0 or 10 nm height, which can unequivocally be identified as edges of flattened vesicles, were reproducibly found be repositioning finder grids in the STM after suitable areas were identified in the STEM. As grids had to be transferred from STEM to STM where they were scanned at ambient pressure, porin membranes were dried in air. Therefore, the lack of distinct, regular surface modulations in STM micrographs of porin-phospholipid lattices is rather due to preparation artifacts than due to instrumental limitation of the STM. Preferably, tunneling microscopy should be done within the electron microscope in order to permit the observation of freeze-dried biological samples, which are expected to exhibit less damaged surfaces [7]. Such a system is presently being developed in our laboratory. Other surface lattices or membranes could be studied in the STM as well. One lattice of particular interest is the purple membrane, because its structure is being analysed at near atomic resolution [15] and because very large, highly regular membranes are available.

Acknowledgments The authors are greatful to Ms. A. Hardmeyer and M. Regenass for producing porin vesicles, and to Ms. H. Frefel, Ms. M. Steiner and Ms. M. Zoller for skillful photographic work. Financial support by the Swiss National Science Foundation (grant 3.251-0.82 to A.E.) and by the “Kommission zur Forderung der Wissenschaftlichen Forschung” to H.-J.G. is greatly acknowledged.

References [1] [2] [3] [4] [S] [6] [7] [8J [9] IlO] [ll] 1121 [13] [14] [15]

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