Scanning tunneling microscopy of planar biomembranes

Ultramicroscopy North-Holland

117

33 (1990) 117-126

SCANNING

TUNNELING

K.A. FISHER,

MICROSCOPY

OF PLANAR BIOMEMBRANES

K.C. YANAGIMOTO

Department of Biochemistry CA 94143-0130, USA

S.L. WHITFIELD,

and Biophysics, and Cardiovascular

R.E. THOMSON,

Research Institute,

M.G.L. GUSTAFSSON

Department of Physics, and Center for Advanced Materials, CA 94720, USA

Universiry of California, San Francisco,

and J. CLARKE

Lawrence Berkeley Laboratory,

University of California, Berkeley,

Received 10 February 1990; in final form 8 March 1990

We combined planar membrane monolayer techniques with scanning tunneling microscopy @TM) to measure the thickness of metal-coated purple membrane (PM) isolated from Halobacterium halobium. Although the metal coating precluded obtaining high-resolution lateral information, it facilitated obtaining high-resolution vertical information. For example, the apparent mean thickness of planar PM and variations in thickness of enzyme-treated PM could be detected and quantified at sub-nanometer resolution.

Abbreviations AFM bR bio-STM ES HOPG HP1 Mh PAGE PLG PLM PM PS PZT SDS STM TEM TMV Z

1. Intro&&ion

Atomic force microscope or microscopy Bacteriorhodopsin Biological STM Membrane extracellular surface Highly oriented pyrolytic graphite Surface protein array of Deinococcus radiodurans Outer cell wall fragment of Mefhanospirillum hungatei Polyacrylamide gel electrophoresis Polylysine-treated glass Polylysine-treated mica Purple membrane Membrane protoplasmic (cytoplasmic) surface Piezoelectric transducer Sodium dodecyl sulfate Scanning tunneling microscope Transmission electron microscope Tobacco mosaic virus Vertical position of STM tip relative to sample plane

03043991/90/$03.50

8 1990 - Elsevier science

Publishers

Our head is round so that our thinking can change directions Francis Picabia

Scanning probe microscopes represent a new approach to investigating the surfaces of physical and biological samples [l-5]. The new microscopes are based on the development of a precise means to control the position of a probe relative to a sample [1,2]. The device for positional control, a piezoelectric transducer, is capable of moving in fractions of %ngstriims, and thus controlling the position of a sensor probe and/or the sample. A variety of probes have been developed to detect electron tunneling, force, thermal variations, ion conductance, electrical conductance, capacitance, light, etc. (see table 1). The principles of the scanning tunneling microscope have been discussed in several reviews [l-8]. In STM, a metal probe or tip that is sharp and electrically conductive is brought close to a conductive surface such that the electron orbitals of

B.V. (North-Holland)

118

K.A. Fisher et al. / STM of planar biomembranes

Table 1 Examples of scanning probe microscopes that have been developed to detect a variety of physical and chemical phenomena at nanometer and atomic resolution (these microscopes are similar in that they control movement of probe, sample, or both by piezoelcctric transducers) Scanning probe microscopes Atomic force Ballistic electron emission Electrostatic force Ion conductance Laser force Magnetic force Near field acoustic Near field capacitance Near field optical Near field thermal Tunneling

the tip and surface overlap. A small potential, usually a few millivolts, applied between the tip and the surface results in a nanoampere-to-picoampere current as an exponential function of distance: the closer the tip to the sample the higher the current. The tip is moved in a raster pattern while feedback electronics either keep the current constant by regulating the distance between the tip and the sample, or keep the distance constant. In the topographic constant current mode, feedback values are displayed on a monitor as single line scans or gray scale images of height variations. Whereas STM has found increasing use in the physical sciences, especially surface science, biological STM has been slow to develop (fig. 1). Biological samples have limited electrical conductivity, are large, and are subject to movement by the STM tip [7,9-111. To improve conductivity, samples are applied to conductive surfaces such as gold or highly oriented pyrolytic graphite and/or are coated with metal. Unfortunately, coating with metal limits the lateral resolution of bio-STM. For samples that are extended in two dimensions, however, such as membrane sheets, it is possible to average bio-STM signals for sample and substrate. The difference between the averaged signals can provide information about membrane thickness at subnanometer

resolution [12]. Thus it is possible to think in terms of vertical resolution; in other words, to add a new quantifiable dimension to high-resolution microscopy.

2. Bidogical applications New tools, especially microscopes, often excite scientists with their potential for answering previously unanswerable questions. Only after the blush of youth has faded do the art and facts (artifacts?) of the technology come to light. Clearly many of the initial biological applications of STM to studies of biomolecules and biostructures fall into the first-blush category, reminiscent of the early days of light microscopy and electron microscopy, when images were given chemical labels despite the absence of supporting analyses. Bio-STM studies have increased steadily since the first images published in the mid-1980s [1,13]. Although samples have ranged from molecules to whole cells, most STM studies of organic and

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YEAR Fig. 1. Plot of abstracts on biological STM versus year. Inset: physics citations during the same period (1980 to July 1989). Bio-SIM abstract number determined from four international STM congresses; physics references from DIALOG databases using the search pattern “scanning (w) tunneling (w) microstop?“. Although these data are neither exhaustive nor especially accurate, they give an idea of relative interest and trend.

K.A. Fisher et al. / STM of planar biomembranes

Table 2 Examples of native and metal-coated organic and biological samples that have been examined by scanning tunneling microscopy (references are representative and not a complete list; also see ref. [9]) Category

Example

Ref.

Molecular

Acetone Amino acids Benxene-CO superlattice Organic conductors Phthalate Phthalocyanines

1141

CdUlOSe

1231 1241

Macromolecular

Supramolecular

Viruses

Cyclodextrin Nucleic acids: DNA (in air) DNA (in water) RNA (in air) RNA (in water) Polypeptides Roteins: Collagen fibrils Fibronectin Hemoglobin Phosphorylase Vicilin Wheat protein Synthetic polymers Bacterial cell walls: D. raa?odurans (HPI) M. hungarei Conductive polymers Detergent monolayers Fatty acid bilayers Langmuir-Blodgett films Liquid crystals Membranes: Reconstituted Native: Cancer cell PM Microtubules Phospholipid bilayers Rotein crystals: Catalase m&-DNA complexes Bacteriophages: @ 29 T4 polyheads T7, fd Tobacco mosaic virus

I151 116,171 1181 1191 [20-221

[24-301 (31-381 I259391 1371 v4,401 [41-451 WI [471 w3.491

1501 1241 151,521 [53-551 156,571 [SS-601 1611 162,631 [23,62&t] [65-671 I68,691 I701 [9,12,71-731 143,741 [41,43,63,70,75,76] I771 [72,78,79] 1131 110,80,81] 1811 158,801

119

biological samples have examined macromolecular and supramolecular assemblies (table 2). Table 2 categorizes some recent bio-STM studies. Although the reference list is not complete and contains abstracts as well as primary references, it is intended to show the range and frequency of samples studied by STM. Relevant to the present paper, a number of studies have been concerned with model membrane systems, as well as native membranes. Model membranes have included Langmuir-Blodgett lipid monolayers and bilayers, and detergent and phospholipid films. Studies of liquid crystals [65-671 and electrically conductive polymers [59] have produced especially striking images. A membrane reconstituted from phospholipid and a protein isolated from E. coli outer membrane, porin, has also been examined [68,69]. The major native membrane studied to date has been purple membrane isolated from Halobacterium halobium [9,12,71-731. Finally two protein arrays isolated from bacterial cell walls - the HP1 layer from Deinococcus radioduruns [53-551

Table 3 The advantages versus uncoated croscopy

and disadvantages (native) samples

Metal-coated samples (stripped or in situ)

Stable (sample anchored) Conductive (metal-metal junction) Height (can be quantified) Theory (good for metal junctions)

of examining metal-coated by scanning tunneling mi-

Uncoated samples (wet or dry) Advantages Native (molecule imaged directly) Hydrated (most like native state) Simple (sample preparation easy) Electrochemistry (is possible)

Disadvantages Vacuum Unstable (sample moved by tip) (sample must be dry or frozen) Metal Nonconductive (coating is required) (tip-sample effects) Heat Height (of metal condensation) (difficult to quantify) Grain Theory (of metal coat) (poor for nonconductors)

120

K.A. Fisher et al. / STM of planar biomembranes

and an outer cell wall fraction from Methanoqirillium hungatei [56,57] - have also been examined. Bio-STM sample preparation can be divided into two categories: techniques to prepare native (uncoated) samples, such as DNA on gold or HOPG substrates, and methods to prepare metalcoated samples, such as membranes on glass or mica substrates. Each method of preparation has its advantages and disadvantages (table 3). In contrast to conventional transmission electron microscopy, bio-STM can image liquid-solid interfaces [82,83] and native samples in water or physiological buffers [31-381. The opportunity to examine fully hydrated biological molecules at nanometer resolution has excited many biological microscopists. However, there are still problems to overcome in imaging hydrated native samples including tip-sample interactions that diminish resolution due to tip displacement of the sample, and difficulties in image interpretation resulting from minimally conductive samples. Coating samples with metal circumvents these problems by physically stabilizing the samples and thus diminishing tip-sample perturbations, and by providing electrical conductivity, thus facilitating image interpretation in topographic terms. Although this paper focuses on metal-coated membrane samples, there have been several published examples of bio-STM studies of uncoated model membranes, lipid films, liquid crystals, and molecules (see table 2 and ref. [9]). The major disadvantages to metal coating are that samples must be first air-dried or frozen, and/or freeze-dried. They are exposed to vacuum, surface contamination, and thermal perturbations associated with the condensation of’ hot metal gases. A further significant disadvantage is that the metal coat, not the sample, is scanned, thus reducing lateral resolution to nanometer dimensions. Nevertheless, convincing images of metalcoated macromolecular assemblies have been published. Examples include complexes of RecA-DNA [72,78,79], freeze-fracture replicas of the layered phospholipid dimyristoyl phosphatidylcholine [75], a paracrystalline protein array, the HP1 layer, isolated from the surface of the bacterium Deinococcus radiodurans [53,54], and phage T4 A-type polyheads [10,80].

3. Planar biomembrane

monolayers

Methods to form microscopically flat monolayers of cells and biomembranes have been developed to facilitate correlating chemical information with microscopic images [9,84-891. The planar membrane monolayer approaches have focused on membranes from two biological systems: cell plasma membranes isolated from red blood cells [90-951 and purple membrane fragments isolated from the halophilic bacterium Halobacterium halobium [96-981. PM is an ideal test sample for scanning probe microscopes [9,12,73]. It has been studied extensively, and is composed of an asymmetric lipid bilayer and a single non-glycosylated protein, bacteriorhodopsin, organized in a paracrystalline lattice [99-1021. Because of the useful and well characterized structural features of PM, several laboratories have described bio-STM studies of fragments of PM [9,12,71-731. Moreover, methods exist [98] for applying PM to cationic surfaces so that it attaches preferentially by either cytoplasmic or extracellular surface (fig. 2). Such PM preparations have been qualitatively and quantitatively evaluated by TEM [96,97]. Membranes that are slowly air-dried will either crack, characteristic of the extracellular surface of the membrane, or pit, characteristic of the cytoplasmic surface of the membrane (fig. 3a) [97]. In contrast, samples rapidly dried with a burst of nitrogen gas or freeze-dried show no gross surface perturbations (fig. 3b). TEM is especially useful for monitoring samples prepared for STM, for example, for measuring the surface coverage of PM applied to different substrates, such as mica, HOPG, and glass, that have been treated with different cations, such as poly-L-lysine and Alcian Blue. Planar, metal-coated PM monolayers are also well suited to quantitative bio-STM studies since membranes are microscopically flat, closely packed, and readily identifiable in both size and shape (fig. 4). We found that PM images are influenced by the planar substrate to which the membrane is attached. For example, glass surfaces are much rougher than mica or HOPG [12,73], and PM attached to glass is similarly rough. Surprisingly, the relative roughness of the sub-

K.A. Fisher et al. / STM of planar biomembranes

APPLY

/Y

SONICATE

WASH

MEMBRANE

DRY

121

6 SHADOW

(20’)

STRIP

6 EXAMINE BY

TEM

Y

WASH

SCAN BY

IN SITU STM

Fig. 2. Planar membrane monolayer preparation. Well-washed purple membrane is suspended in 1OmM buffers, applied for 30 s to polylysine-treated glass (PLG) or mica (PLM) substrates, and washed with buffer for 30 s. Samples are transferred to a beaker in an 80 kHz bath-type sonicator, sonicated for 15 s, washed with 1N NaCl for 30 s, and finally washed with distilled water. &&lowing the beaker during washes removes molecular debris floating at the air-liquid interface. Samples can then be chemically or enzymatically treated, and/or dried by nitrogen burst, or rapidly frozen and lyophilized (freezedried or deep-etched). Spmples are shadowed with thin metal films of Pt-C or Pt-Ir-C in vacua at an angle of 20° for TEM, and coated at an angle of 90° for SIM. TEM replicas are strengthened by carbon evaporation, stripped from the substrate by floating onto diluted HF. cleaned with detergents and bleach, washed, and transferred to TEM grids. Metal-coated STM samples are neither carbon-coated nor stripped from the substrate but scanned in situ.

strate has minimal effect on quantitative studies of the thickness of PM, presumably since the similar rot&tresses of metal-coated substrate and metalcoated membrane cancel out. Our original goal was to image the polypeptide chains of bR exposed at the two surfaces of the membrane. We found, however, that coating PM with metal obscured high-resolution lateral information. Others have discussed similar limitations, for example, in studies of metal-coated and uncoated samples [10,53,54]. Our attention then focused on vertical or membrane thickness measurements. It is well known that the STM is espe-

cially sensitive to vertical displacements, and can measure differences with an accuracy better than 0.01 nm [4,5,7,11]. This suggested that bio-STM was .potentially capable of measuring membrane thickness at heretofore unobtainable resolution.

4. Membrane thickness meamement Using the STM as a morphometric tool required not only reproducible sample preparation but also careful calibration of STM tip movement and evaluation of tip/sample interactions [12,73].

K.A. Fisher et 01. / STM

ofplanar biomembrones

Fig. 3. TEM images of purple membranes (PM) attached to PLG and either slowly air-dried (a), or rapidly nitrogen-dried (b), and shadowed with Pt-C. (a) PM applied for 30 s at pH 5 to facilitate random adsorption, then washed with distilled water. Slow air drying produces artifacts of either cracks or pits that identify membrane surfaces. Cracked surfaces represent the extracellular surfaces (ES) of PM, pitted surfaces the cytoplasmic (protoplasmic) surface (PS). (b) PM applied as in (a), and dried with a burst of nitrogen gas delivered from an air gun. PM shape and flatness make it an ideal sample for STM studies. Shadow direction bottom to top. Bars = 0.5 urn.

Fig. 4. STM image of PM attached to PLM, rapidly frozen and deep-etched. Micrograph shows six PM fragments. The Pt-IrC coated sample was scanned in situ, in air, at ambient temperature using a cut Pt-Bh tip operating at a tunneling current of 3 nA and bias voltage of 1.0 V, sample negative. STM data were computer-processed to remove I drift, and presented as a top-view gray scale image where black = mica substrate and white = highest point above substrate. Bar = 0.2 Pm.

We found that dynamic calibration of tip movement using laser interferometry was more reproducible than static calibration. For bio-STM thickness measurements, however, a better method may be to scan simultaneously the unknown and standards with known height distributions, such as size-selected colloidal gold particles. Adsorbatemediated surface deformation of samples lying on layered surfaces such as mica or HOPG [103] is an example of a tip-sample interaction of potential significance to membrane thickness determination (fig. 5). Unless prepared and examined under ultrahigh vacuum conditions, both tips and samples are contaminated with water and/or organic adsorbates. Contaminants cause the tip to press through the adsorbate to achieve electron tunneling, and the resultant force on the substrate deforms it. A substrate such as HOPG can be significantly deformed in response to the downward force of a contaminated tip being scanned in air [103]. As the tip retracts, the surface follows it at constant electron current, thus producing a height that is anomalously large. In our study of PM thickness, we measured deformation curves for metal-coated glass, mica, and membrane, and

RA. Fisher et al. / STM of pkmar biomembranes IDEAL

REAL

Fig. 5. Cartoon illustrating STh4 tip problems. The ideal tip (nonexistent) would consist of a single uncontaminated atom on a stable atomic column. Real tips, however, are often multiple (producing “ghost” images), large (reducing re-solution) and contaminated (producing surface deformations).

found them to be similar. Importantly, we found similar thickness values for metal-coated membranes independent of the substrate glass or mica. The procedure used for measuring membrane thickness was analogous to determining the spectral properties of a highly scattering sample by difference spectroscopy between sample and reference. The sample in this case was metal-coated membrane, and the reference, metal-coated substrate adjacent to the membrane. To quantify thickness, we verified that both membrane and substrate were identical with respect to variations in metal grain size or shape, conductivity, surface tip-surface interactions (steric, contamination, mechanical), signal noise, and signal processing (filtering, smoothing). Under these conditions the variables due to chemical and physical properties and signal processing cancelled out. Because accuracy was further improved when large areas of membrane and substrate were compared, large data sets were collected, and a plane calculated for each data set by least-squares a&lyses. Subtracting the substrate plane from the membrane surface plane produced a voltage difference that could be converted into membrane thickness given the calibrated displacement of the PZT. Because STM had not previously been used to quantify the thickness of PM we measured thickness on different substrates, glass and mica, and under two drying methods, nitrogen- and freeze-

123

drying, and further verified thicknesses independently by TEM measurements of shadowed samples. There was a remarkable quantitative consistency among different sample-preparation techniques and between the two physically different modes of microscopy. The pooled mean sample values of bio-STM measurements of PM on glass (4.6 nm) and mica (4.6 nm) and TEM measurements.on glass (4.8 nm) and mica (4.5 nm) gave an overall mean of 4.63 nm, with a standard deviation of 0.13 nm.

5. Measuring changes in membrane tkkness We also asked the question “Is it possible to measure changes in the thickness of membranes after enzymatic modification?” Planar monolayers were again useful for such analyses, since enzymetreated membrane monolayers could be analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis to evaluate the effect of the enzyme on attached membranes [12]. Oriented planar monolayers of PM were treated with the protease papain, known to remove about 22 amino acids from the carboxyl-terminus of bR, located on the cytoplasmic side of the membrane. Treated and untreated PM were examined by SDS-PAGE, TEM, and bio-STM. The pooled mean STM thickness value of papain-treated PM on glass (5.4 nm) and mica (4.8 nm) and TEM measurements of papain-cleaved PM on glass (5.7 nm) and mica (5.3 nm) gave an overall mean of 5.3 nm, with a standard deviation of 0.4 nm. Thus the apparent mean thickness of enzyme-cleaved PM (5.3 nm) was consistently 0.7 nm greater than untreated PM (4.6 nm). The word “apparent” in the phrase “apparent thickness” should be emphasized. Removal of 1722 amino acids from the surface of the membrane would be expected to decrease rather than increase the thickness of the membrane. Although there are several plausible explanations for the unexpected observation of apparent increase in thickness, it is most likely that the increase is a function of the degree of attachment and/or interaction between PM and the substrate [12]. Regardless of the explanation, the important point is that

124

K.A. Fisher et aL / STM ojplanar

bio-STM can detect sub-nanometer differences in membrane thickness, potentially more accurately than can conventional TEM and at significantly higher resolution than SEM. Atomic force microscopy has recently been used to examine biological membranes including PM [104,105]. Although the first published AFM image of PM was of low resolution, the authors stated that one of the two membrane surfaces showed “protruding disc-shaped features forming a hexagonal lattice with about 6 nm center to center spacing” [104]. A more recent AFM study showed clear PM images and reported periodic orders of 8.7 nm on glass and 11.6 nm on mica [105]. Although these latter values were consistently larger than the 6.2 nm spacing measured by electron diffraction of bulk samples, the AFM studies point to the utility of the PM system for evaluating the quantitative characteristics of the new microscopes.

6. Conchrslona Scanning probe microscopes represent a revolution in microscopy. Their uniqueness is based on utilization of a PZT that can control probe movements with atomic precision. A wide variety of probes have been designed to measure physical properties with nanometer accuracy. In addition, since probe signals are routinely obtained digitally, images and information can be digitally processed, a curse or a blessing depending on one’s point of view. As a field, bio-STM has only just begun. Much more effort is needed before its potential is fully realized. Biological samples are chemically and physically heterogeneous and when scanned on layered substrates produce images that are difficult to interpret strictly in topographic terms. Woefully lacking, and reminiscent of the earliest days of TEM, are means to gain direct chemical information from the images. Chemical markers analogous to colloidal gold labels for TEM and STM need to be developed for STM. Altematively, further development of spectroscopic and other modes of scanning may be advantageous. The atomic resolution capabilities of the STM

biomembrmes

have yet to be established for bio-STM. Although metal coating techniques fix the sample to the substrate and provide electrical conductivity, the metal obscures specimen detail at the highest levels of resolution. Nevertheless, if one thinks in terms of vertical rather than lateral resolution, the metal coating facilitates application of bio-STM to measurements of sample thickness, i.e., it literally extends our ability to gain morphometric data to another dimension. Where is the field of biological scanning probe microscopy going? The limitations of bio-STM have led some researchers to focus on other scanning probe microscopes, such as the AFM. And for both bio-STM and bio-AFM, one quest is the same: higher resolution. The present resolution for biological samples is in the nanometer range, whereas the resolution for electrically conductive physical samples has been shown to be in the &ngstriim range. Several laboratories have begun to develop cryogenic scanning probe microscopes in an attempt to improve resolution with biological samples. The expectation is that liquid nitrogen or liquid helium temperatures will immobilize the sample and diminish thermal drift of the PZT, two factors thought to limit biological sample resolution.

Adcnowkdgements We thank Mrs. Eleanor Crump for editorial help. This work was supported by a grant from the Public Health Service, NIDA No. DA 05043 (K.A.F.), and by the Director, Office of Energy Research, Office of Basic Energy Science, Materials Science Division of the US Department of Energy, under contract No. DE-AC03-76FOOO98 (J.C.).

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