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
PUTSICS cIT4mIons YEAR
RUM
‘80-‘86 ‘87-‘88 ‘8&‘89
562 693 653
1.898
nxAL
; I I
t
/
,/’ .A
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
PUTSICS cIT4mIons YEAR
RUM
‘80-‘86 ‘87-‘88 ‘8&‘89
562 693 653
1.898
nxAL
; I I
t
/
,/’ .A
