High resolution imaging of native biological sample surfaces Andreas Engel

279
High resolution imaging of native biological sample surfaces
using scanning probe microscopy
Andreas Engel∗†, Cora-Ann Schoenenberger∗ and Daniel J Muller
¨ ∗‡
The possibility of acquiring high resolution topographs
using scanning probe microscopes under physiological
conditions allows the observation of biomolecules at work.
Progress has recently been made in imaging protein–DNA
complexes, individual oligomers and protein arrays. Scanning
probe microscopes are now tools that complement X-ray
crystallography and electron microscopy.
Addresses
∗ME Muller
¨
Institute for Structural Biology, Biozentrum,
Klingelbergstr 70, CH-4056 Basel, Switzerland
†e-mail: [email protected]
‡Forschungszentrum Julich,
¨
IBI-2: Structural Biology,
D-52425 Julich,
¨
Germany
Current Opinion in Structural Biology 1997, 7:279–284
Electronic identifier: 0959-440X-007-00279
Scanning near field optical microscopes (SNOMs) measure
the local optical properties of a sample using an aperture as
a probe. Significant progress in the underlying technology
has lead to the detection of single fluorophores [3], but
the resolution achieved is at least an order of magnitude
less than that of the STM. For the latter, however, the
sample must be electrically conducting, which precludes
biological structures as they are notorious insulators. High
resolution scanning probe microscopy of biological samples
is primarily achieved using the AFM. This instrument
also allows samples to be scanned in solution, opening
up the fascinating possibility of observing biomolecules at
work. Excellent reviews are now available that illustrate
various applications of the AFM to biological research
[4–7]. This review concentrates, with a single exception,
on high resolution images acquired with the AFM.
 Current Biology Ltd ISSN 0959-440X
Abbreviations
2D
two-dimensional
3D
three-dimensional
AFM
atomic force microscope
SNOM scanning near field optical microscope
STM
scanning tunneling microscope
Introduction
Invented more than a decade ago [1,2], scanning probe
microscopes are powerful tools to study the surfaces of
solids in vacuum, gas or liquid at atomic-scale resolution.
A small probe (with an atomic size of up to a few tens
of nanometers) is scanned over the sample surface and
the signal produced by the sample–probe interaction is
acquired. The height (z) of the probe relative to the
sample can either be kept constant during the raster-scan
in the (x,y)-plane, or it can be controlled by a servo system
to keep the interaction signal constant. In the latter mode,
the surface corrugations are contoured.
To achieve high resolution, the decay length of the
sample–probe interaction must be small. The tunneling
current between the probe tip and the conducting sample
typically changes by a factor of ten when the tip–sample
distance changes by 1 A˚. Whereas van der Waals and
electrostatic static interactions between the tip and
the sample extend over several nanometers, shell–shell
repulsion forces rise very sharply (by a factor >10 A˚−1)
when the tip is in contact with the sample surface. Atomic
resolution, therefore, has been obtained using scanning
tunneling microscopes (STMs) and using atomic force
microscopes (AFMs).
Instrumentation
The heart of the AFM is a pyramid-shaped stylus (with an
apex radius of 2–10 nm) mounted on a flexible cantilever
(spring constant k = 0.1–1 Nm−1). A laser beam reflected
from the back of this lever onto a multifaceted photo diode
allows deflections of < 1 nm to be detected (Fig. 1). Hence,
an AFM may be operated in contact mode with a force
applied to the stylus of about 10−10 N. The resonance
frequency of the cantilever sets a limit to the speed for
contouring a surface; this speed is slower in liquids than
in vacuum as result of damping [8]. A piezo scanner is
actuated to translate the sample relative to the tip in x,
y and z directions. The corresponding signal is provided
by the computer that drives the microscope and acquires
the images. Multiple images may be collected in parallel
by taking advantage of different imaging modes.
A useful image, akin to differential interference contrast
in light microscopy, is generated by the error or deflection
signal [9]. When flat samples are scanned slowly, the
piezo scanner is able to follow the surface corrugation
precisely, thus counteracting the cantilever deflection. On
corrugated samples, however, the servo speed is too slow
and a deflection is inevitable. This deflection signal is
valuable for low magnification work, in which the AFM
is operated with a high scan speed. To monitor the
absolute height information, the z-signal (or height signal)
driving the piezo scanner is recorded simultaneously. In
addition, it is possible to reconstruct the topography from
the deflection signal, approximating the latter as the
first derivative of the height signal along the fast scan
direction [10••].
280
Macromolecular assemblages
Figure 1
Physical constraints of scanning probe microscopy. (a) Simple geometric model to estimate the resolution. A spherical particle of diameter
D (shown on the left) exhibits a full width at half maximum (FWHM =2√ (RD + D2/4) when imaged with a spherical tip of radius R. The
true height of the particle is measured. An array of spherical particles scanned with the same tip (shown on the right) yields a corrugation
amplitude, h ≈ (D/8R)1/f, using the spatial frequency of the array, f = 1/D. Thus, a scanning probe microscope has approximately an 1/f transfer
function. This is a qualitative description, however, as the imaging process is strictly nonlinear, as illustrated by the small gray sphere on the
left that does not produce a signal when close to the larger particle but that would be seen when isolated. Such hard sphere models do
not take sample flexibility and long range tip–sample interaction into account. (b) Interactions between substrate and sample in electrolyte
solutions. The interactions are symmetric to those between the sample and the tip, unless the sample is covalently bound to the support.
Immobilization may therefore require different buffer conditions than imaging. For adsorption, the Debye layer thickness λD needs to be minimized
(λD =0.304/√ [ec] nm for monovalent electrolyte concentrations, ec; [34•]) to allow sample adsorption by van der Waals attraction [14]. For
imaging, however, the electrolyte should be selected to equalize electrostatic repulsion and van der Waals attraction. For large sheets, it is
possible to change the buffer without detachment of the sample.
Substrate–sample and sample–stylus
interactions
Operating the AFM in contact mode induces friction; in
order to withstand friction forces, samples need to be
immobilized. Large structures adhere well to substrates or
can simply be pushed into small holes. Small structures
(e.g. single protein complexes, thin filaments), however,
exhibit only a small contact area for interaction with the
substrate. Protocols have been developed to covalently
bind biomolecules to the support [11,12]. Alternatively,
friction forces can be minimized by oscillating the
cantilever vertically; using this ‘tapping mode’ [13]
weakly adsorbed samples can be observed, albeit at
somewhat reduced resolution. Samples may firmly bind to
freshly cleaved mica that exposes a weakly (negatively)
charged, chemically inert surface. In this case, the sum
of electrostatic repulsion and van der Waals attraction
determines whether samples adsorb. Electrostatic forces
depend on the surface-charge density of sample and
substrate, and on the electrolytes in the buffer solution.
Sample adsorption is thus controlled by the nature and
concentration of electrolytes, as recently demonstrated
using a variety of samples [14].
Interestingly, the interactions between sample and stylus
involve to a large extent the same forces that occur
between sample and support. Only the very tip that is in
contact with the surface atoms will sense the shell–shell
repulsion. It is this short range interaction that confers
high resolution structural information. The symmetry in
the interactions between the substrate, sample and stylus
implies that not necessarily the same buffers should be
used for adsorption as for imaging. In addition, height
measurements are profoundly affected by the electrolytes,
as the surface-charge density of the sample and the
support are, in general, different (DJ Muller,
¨
A Engel,
unpublished data).
It is important to note that
orders of magnitudes smaller
that occur when samples are
hydrophilic surface is covered
molecules [15,16].
these forces are several
than the capillary forces
observed in air, as every
by several layers of water
High resolution imaging
Filamentous structures
DNA was an attractive sample with which to test the
capability of the STM in the early days of scanning
probe microscopy. The first results fostered hopes that it
would become possible to sequence DNA using an STM.
This goal has not been achieved, and many results have
subsequently been identified as artefacts. Nevertheless,
double-stranded DNA molecules adsorbed to mica can
be imaged in a humid atmosphere at high resolution,
using an STM capable of detecting tunneling currents of
10−12 Amp (Fig. 2a) [16].
Clear images of DNA can be obtained at ambient
pressure using the AFM when it is flooded with dry
nitrogen to minimize capillary forces, or when the tapping
mode is used [7]. For the imaging of DNA and native
protein–DNA complexes in buffer solution, much effort
has been invested in the immobilization of DNA on the
High resolution imaging of native biological sample surfaces Engel, Schoenenberger and Muller
¨
281
Figure 2
Scanning probe microscopy of DNA. (a) Plasmid DNA (pUC 18) on mica imaged by an STM at high resolution (inset) in a humidity chamber (at
66% relative humidity) [16]. The thin water layer coating on the sample has a thickness of ≈ 0.4 nm and exhibits a high conductivity of up to five
magnitudes greater than bulk water. The water layer, therefore, enables the imaging process with an STM. (b) AFM image of double stranded l
HindIII DNA attached onto mica [17]. To reduce the capillary forces that occur in air, the image is recorded in propanol. (c) The same molecule
recorded after one minute demonstrates the reproducibility of the imaging process. Gray shades from dark to bright correspond to a vertical
distance of 2.5 nm in (a), 1.8 nm in the inset of (a), and 0.5 nm in (b) and (c). STM images courtesy of R Guckenberger, Max-Planck Institute,
Martinsried, Germany; AFM images courtesy of H Hansma, University of California, Santa Barbara, USA.
Figure 3
Atomic force microscopy of single protein complexes. (a) CryoAFM image of human IgG monitored at 85K in a vacuum [18•]. The characteristic
Y shape of IgG is clearly visible. After adsorption onto mica, the sample is dried in a nitrogen stream and transported into the AFM chamber.
(b) Surface plot of an AFM image of GroES oligomers imaged in solution [19••]. After adsorption onto mica, the molecules are fixed with 2%
glutaraldehyde. The fine features at the top of the dome reveal seven subunits, which correlates well with the atomic structure of GroES [35].
Gray shades from dark to bright correspond to a vertical distance of 6.5 nm in (a), and 3.5 nm in (b). Images courtesy of J Mou and Z Shao,
University of Virginia, Charlottesville, USA.
substrate, but improved images have been obtained by
employing the tapping mode (Fig. 2b) [17].
Oligomeric complexes
Antibodies are among the most interesting biomolecules
that are studied by scanning probe microscopy, because
imaging these biomolecules at high resolution under
physiological conditions opens an important field of
applications. The results have been rather disappointing.
As antibodies are relatively bulky and flexible structures,
they are seen mainly as blobs without substructure. The
immobilization of IgGs using cooling techniques has led
to images that reveal the Y-shaped molecules (Fig. 3a)
[18•]. Higher resolution can be obtained on less corrugated
282
Macromolecular assemblages
Figure 4
Regular protein arrays
The tightest molecular packing is achieved with regular
protein arrays and the highest resolution images have
been acquired on such 2D crystals [21]. The contours
determined with the AFM have been compared with 3D
structural information from electron microscopy [10••,22]
and X-ray crystallography [23••]. These experiments have
confirmed that the data acquired using the AFM under
¨
native conditions agree to within a few Angstroms
with
data obtained by other methods. A thoroughly studied
example is the Escherichia coli porin OmpF (Fig. 4).
AFM image of OmpF. The periplasmic surface of OmpF trimers
reconstituted into a rectangular 2D crystal is a suitable sample with
which to study the reliability of the AFM. The crystalline sheets adsorb
readily to freshly cleaved mica in 300 mM KCl, 10 mM Tris-HCl,
pH 7.4. The topograph shown has been recorded on a Nanoscope III,
using a Olympus cantilever (k = 0.1 N m−1), applying a force of 100 pN
to the stylus. The trimers exhibit a tripartite protrusion consisting of a
triangular mass with an indentation about the threefold axis and three
arms. This structure protrudes by 0.5 nm from the bilayer surface
and separates the three elliptical channels. The striking feature of
this topograph is its signal-to-noise ratio: the substructure of each
trimer is clearly resolved. In some instances, damaged trimers are
distinct (indicated by arrows). The tip used to acquire this image has
an elongated apex, which leads to a minor astigmatism. A rectangular
unit cell (a = 13.8 nm, b = 7.9 nm) is marked; gray shades from dark to
bright correspond to a vertical distance of 1.5 nm.
Surface topographies of purple membranes have revealed
discrepancies with respect to the initial atomic model of
bacteriorhodopsin in that the cytoplasmic loop connecting
helices C and D exhibited the same height as loop
AB, and that loop EF was not visible (Fig. 5a) [24]. It
has subsequently been observed that loop EF becomes
visible when the force applied to the stylus is < 200 pN
(Fig. 5b) [25••]. In addition, the new atomic model of
bacteriorhodopsin reveals that the AB and DC loops
are at comparable heights [26]. Purple membranes are,
therefore, a suitable sample to test the quality of an
AFM, the preparation protocol, and the operator’s skill:
if loop EF can be seen, the overall system is tuned
to detect rather delicate protein structures. The highest
resolution achieved is with samples that have a corrugation
amplitude < 0.5 nm, such as the extracellular surface of
purple membranes (Fig. 5c) [21].
High resolution imaging is not restricted to 2D crystals.
Recently, promising data have been recorded on 3D
protein crystals allowing the study of crystal packing and
its defects [27•].
Conclusions
biomolecules because they are less flexible and can be
contoured precisely by the probe tip. When the stability
is further improved by glutaraldehyde fixation, single
oligomers can be imaged at 1 nm resolution (Fig. 3b)
[19••]. Although these images exhibit superb resolution,
the preparation steps required for a sample impeach
observation of biological activities.
Densely packed proteins
The geometry of the probe tip restricts access to the
topmost fraction of a spherical or cylindrical sample.
Tightly packed oligomers appear to be an ideal sample:
proteins laterally support each other, hiding surfaces that
the tip cannot reach in any case. Membrane proteins can
often be reconstituted in lipid bilayers at high density or
can be inserted in supported monolayers. Such examples
include various toxins, whose surface features have been
resolved with the AFM [20].
Scanning probe microscopes are tools that entail interesting applications for structural biologists; in particular,
the AFM has demonstrated its capability of acquiring
surface topographies with a lateral resolution of 0.5–1 nm
and a vertical resolution of 0.1–0.2 nm when the biological
sample is in a buffer solution. Conformational changes can
now be detected at subnanometer resolution, which opens
a new avenue to monitor the relationship between structure and function of biomolecules [28,29•]. Such exciting
observations suggest that structure–function determinations may become a major application of AFM. The high
sensitivity of force measurements has initiated an ever
growing variety of experiments that tackle the question of
interaction forces between macromolecules [30–32,33••].
The knowledge gained from this work will help to increase
the sensitivity of the instruments to allow samples to be
scanned at even lower forces. Combined with the progress
in understanding the tip–sample interactions [15,34•],
these developments are expected to increase not only
the resolution, but also the reproducibility and ease of
scanning probe microscope operation.
High resolution imaging of native biological sample surfaces Engel, Schoenenberger and Muller
¨
Figure 5
283
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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High resolution topographs of native purple membrane adsorbed onto
mica in buffer solution. (a) Cytoplasmic surface imaged at 300 pN.
The donut shaped structures represent the bacteriorhodopsin trimers.
(b) When imaged at about 150 pN the structure observed in (a)
transforms into units with three pronounced protrusions at their
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Extracellular surface of purple membrane imaged at a resolution of
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•
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19.
••
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••
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•
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•
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¨
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••
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••
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35.
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•
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