Molecular microscopy
Picture perfect
pentacene
Advances in microscopy are letting us see not just atoms but the chemical bonds in
between them. James Mitchell Crow takes a closer look
Carbon must be one of the most
photogenic members of the
periodic table. Not just in its
diamond form, either – over the
years, chemists have crafted it
into some pretty iconic structures.
Buckyballs and nanotubes are just
some of the more recent examples
of hydrocarbons that have enjoyed
the limelight. Add a smattering of
heteroatoms and structures such
as the famous double helix of DNA,
perhaps the most famous molecule
of all, become possible.
But pentacene? Until recently,
this unassuming polycyclic aromatic
certainly wouldn’t have fallen into
that category. In mid-2009, all that
changed, thanks to a single image
captured by a team at IBM Research
in Zurich, Switzerland.1 That year
the team published a remarkable
snapshot of a single pentacene
molecule, an atomic force microscopy
(AFM) image that captured a level of
detail unprecedented in an organic
compound, clearly showing every
atom and every bond. Strikingly, the
molecule looked exactly like you’d
draw it in a text book.
46 | Chemistry World | January 2011
In short
 In the last three
years, breakthroughs in
microscopy have brought
chemical bonds into view
for the first time
 Adding CO to the
tip of an atomic force
microscope produced
a textbook image of
pentacene
 Putting H2 under the tip
of a scanning tunnelling
microscope could reveal
supramolecular H-bonds
 The techniques
can be applied to real
problems like structure
determination
‘That image of pentacene is
amazing – particularly to chemists,
I think,’ says Neil Champness, a
supramolecular chemist at the
University of Nottingham, UK.
‘What’s most striking is actually
seeing the bonds. We’ve seen
images of atoms before – there’s
the classic image of ‘IBM’ spelled
out in xenon atoms – but seeing
bonds is extraordinary. In some
ways chemists play with atoms,
but really what we spend our lives
doing is forming and breaking bonds.
Actually seeing that side of it is really
pretty astounding.’
Probing particles
Of course, there
are other ways
to detect single
molecules –
such as by
fluorescence
spectroscopy,
for example.
Atomic force microscopes Working
use sharp tips rather than with single
molecules
light to feel the shape of
reveals
objects
reaction details usually lost to
the averaging effects inherent
in conventional techniques
that probe molecules en masse.
Single molecule fluorescence
spectroscopy provides that detail
in real time, an advantage that has
been exploited by Peng Chen and
colleagues at Cornell University in
New York, US.
Chen recently used the technique
to study the catalytic properties
of gold nanoparticles, using a
reaction he designed to turn a
non-fluorescent reactant into a
detectable fluorescent
product. The
team showed
not only that
the catalytic
properties
of each
nanoparticle
differ according
to its structure,
but that every
individual gold
nanoparticles’
structure is also in
a state of constant
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JAMES KING-HOLMES / SCIENCE PHOTO LIBRARY
flux, meaning that their catalytic
activity varies over time.2 ‘Before
our work, people didn’t even know
how to think about the temporal
behaviour of a single nanoparticle
in terms of its catalytic properties.
We were able to measure it directly
and quantify these time-dependent
behaviours,’ says Chen.
As well as gaining fundamental
knowledge that could help guide
future nanocatalyst design, the
team also work in bioinorganic
chemistry. Metals are essential for
many biological processes – for
example, many proteins need metal
ions to function – but these ions
are also toxic if left floating free,
so the body uses proteins called
metallochaperones to move them
from place to place. Chen’s team are
using single molecule fluorescence
techniques to probe the interactions
between metallochaperones and
the proteins that they are delivering
the metal ions to, which bind
together surprisingly weakly. ‘These
interactions are essential, we’re
trying to quantify them using our
technique.’
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Smile please
However, while such techniques
allow us to indirectly follow the
fate of single molecules, they don’t
provide a picture of the molecule
itself, which is why the image of
pentacene is so startling. The IBM
team didn’t set out to find a new
technique for imaging molecules,
says team leader Leo Gross. They
came across the phenomenon by
accident while developing ways to
study molecular electronics and
single electron devices on surfaces.
About five years ago, when Gross
joined IBM, the team were using
scanning tunnelling microscopy
(STM), which images surfaces via
a current of electrons flowing from
the microscope tip to the sample.
However, as the team are looking to
study increasingly thick insulating
films, STM has become a less suitable
technique, so about two years ago
Gross began to use AFM, which
detects changing forces as the tip is
dragged across the surface. It was
while using AFM that he suddenly
began to see sample molecules with
incredible resolution.
Modern microscopes
can work at high vacuum
and low temperature to
improve resolution
‘We chemists
spend our
lives forming
and breaking
bonds. Actually
seeing that is
really pretty
astounding.’
The trick to that jump in resolution
was to create an extremely sharp
AFM tip by adding a CO molecule to
the end of it. ‘By chance we had the
CO tip one time, and suddenly saw
this incredible resolution. Of course
then we tried to improve on it, and
reproduce it,’ he says.
Gross might have captured his
image of pentacene using AFM, but
it was his experience with STM that
lead to the breakthrough. ‘From
our work with STM we have a lot of
knowledge on atomic manipulation,
pulling and pushing molecules on
a surface but also picking up single
atoms or molecules with the tip,’
he says. The CO not only gives a
very sharp tip, it also allows it to
be brought very close to the target
molecule without accidentally
picking it up or moving it, which
would blur the image.
However, it turns out that STM can
also be adapted to capture equally
impressive images – and that perhaps
an image of tetracene captured using
STM in 2008 deserves just as much
fame as Gross’s pentacene picture
captured the following year.3 The shot
Chemistry World | January 2011 | 47
NATURE CHEMISTRY
SCIENCE / IBM RESEARCH, ZURICH, SWITZERLAND
Molecular microscopy
of tetracene looks like a photographic
negative of the pentacene images
Gross produced, the bonds forming
dark lines across a bright background.
Again, the trick to boosting
sensitivity is to capture a molecule
between the tip and the sample –
this time hydrogen or deuterium,
hence the technique has been
dubbed scanning tunnelling
hydrogen microscopy, or STHM,
by its discoverers Stefan Tautz and
colleagues at the Jülich Research
Centre in Germany. As for the
process itself, it was discovered by
happy accident.
‘When we first saw these images
we were absolutely stunned,’ says
Tautz. Initially the effect would
appear and then suddenly disappear
without warning. ‘At the time we
did not know that it was hydrogen
causing the effect, so we had to
systematically try to see what
was happening. Now we can very
reliably reproduce these imaging
conditions.’
The two techniques might sound
similar, but in practical terms work
rather differently. Whereas Gross’s
CO molecule is chemically bonded to
the AFM tip, the hydrogen molecules
Tautz uses simply physisorb onto
the sample surface. As the STM
tip is brought close to the sample,
a single hydrogen molecule gets
trapped between the two, generating
repulsive forces as it is squeezed
between tip and sample. As the tip
is scanned across the surface, these
repulsive forces change according
to the topography of the sample,
bringing the hydrogen slightly closer
to the tip over raised structures.
‘Then this affect called Pauli
repulsion sets in,’ Tautz explains.
‘If you press the hydrogen molecule
into the electron density of the tip,
the electrons are pushed away from
the tip apex, changing the size of the
tunnelling current.’ So the hydrogen
molecule turns the STM tip into a
nanoscopic force sensor, but also
acts as a transducer, converting this
signal into a changing current that
the STM can detect.4
Not just pretty pictures
The images produced by these
techniques might be beautiful,
but could they also be useful?
Undoubtedly so, say Marcel Jaspars
and Rainer Ebel, natural product
chemists from the University of
Aberdeen in the UK.
‘My wife spotted the story about
pentacene, and she pointed it out
to me,’ says Jaspars. ‘The next day I
read the paper in Science, and there
48 | Chemistry World | January 2011
are some sentences in it saying that
the technique was able to find bond
lengths, bond order, and the position
of all the atoms – and I thought that
sounded very much like structure
determination of the kind we do.
‘So I contacted Leo [Gross] saying
that I had a problem – a molecule
that we think is flat, for which
we have been unable to solve the
structure – and asking if he would be
willing to take a look at it. Leo was
willing to try it – the idea that this
might be possible was very exciting
for all of us.’
The compound in question,
cephalandole A, is produced by a
pressure-tolerant bacterium called
Dermacoccus abyssi, which was
Adding carbon monoxide
to the tip of an AFM stops
it nudging molecules
around and allows bonds
between atoms to be seen
Seeing the shape of a
molecule can help solve
riddles like the structure
of cephalandole A
isolated from sediment collected
from the Mariana Trench of the
Pacific Ocean, which at 11km below
sea level is the deepest place on
Earth. The team had been unable
to grow crystals of the compound,
so hadn’t been able to identify the
structure by x-ray crystallography.
The AFM technique immediately
helped solve the structure.5
‘We had some initial structure
proposals,’ says Ebel, ‘but seeing
even the initial [AFM] picture told
us immediately that we were going
completely the wrong way, and we
could very quickly come up with
the right conclusion regarding our
molecule – it was an absolutely
fantastic experience.’
Admittedly, both STHM and
CO-sharpened AFM have a
key limitation – they are only
suitable for planar molecules. ‘At
the moment we can’t get three
dimensional data – we cannot look
below the first layer of atoms,’
says Gross. It is possible in certain
circumstances to flip a molecule
over to view its different faces one
by one, as has been done for C60,
for example, but it isn’t possible to
look inside the molecule with high
resolution.
However, even if it remains
largely limited to planar molecules
such as cephalandole A, that still
leaves a big chunk of molecular
space for which the technique
could become invaluable, because
it is the flat molecules that are
often hardest to assign. ‘Looking
through natural products that had
been misassigned in the past, a
large number of those were either
primarily flat, or had flat elements
to them,’ says Jaspars. ‘Flat
molecules tend to have very few
carbon-hydrogen bonds, and these
are the elements you need to get a
good NMR structure,’ he adds.
Is that a hydrogen bond I see before me?
AFM and STHM might be able to
produce stunning images of the
covalent bonds that hold together
organic molecules – but are these the
only kind of bond that you can see?
Almost certainly not, say the teams
who have developed the techniques
– both have seen structures in their
images that look strikingly like
hydrogen bonds.
Tautz says that he sees areas of
contrast in his images between
neighbouring molecules that appear
to coincide exactly with where
you would expect to see hydrogen
bonds.6 However, whether this
really does show a hydrogen bond
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References
1 L Gross et al, Science, 2009, 325, 1110
(DOI: 10.1126/science.1176210)
2 W Xu, J S Kong and P Chen, Phys. Chem.
Chem. Phys., 2009, 11, 2767 (DOI: 10.1039/
b820052a)
3 R Temirov et al, New J. Phys., 2008,
10, 053012 (DOI:10.1088/13672630/10/5/053012)
4 C Weiss et al, Phys. Rev. Lett., 2010, 105,
086103 (DOI: 10.1103/PhysRevlett.105.086103)
5 L Gross et al, Nat. Chem., 2010, 2, 821
(DOI: 10.1038/nchem.765)
6 C Weiss et al, J. Am. Chem. Soc., 2010, 132,
11864 (DOI: 10.1021/ja104332t)
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James Mitchell Crow is a science
writer based in Melbourne, Australia
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
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JOHN DALTON
A luxury toy?
Whether these techniques will ever
become routine for such applications
is difficult to say, but progress so far
has been encouraging, says Gross.
‘Just two years ago we needed
weeks or months to get such a
nice image, and now we can
reproducibly do it within a
day. With STHM as well,
I feel there’s a lot of
progress in the field.
But then in the end you
don’t know how this
might evolve.’
One particularly
useful extension to the
technique would be to combine
imaging with spectroscopy, for
example to identify heteroatoms in
the structure – a process that should
Things have moved on
be perfectly possible to do, as AFM
fundamental
a lot since the original
questions about what
atomic force microscope can already be used for spectroscopy.
‘By NMR, if we want to determine the
hydrogen bonds are doing in a surface was built in 1985
position of a bromine or chlorine, for
environment, and to be honest we
example, we can do it only indirectly
don’t know. Maybe we’re missing
by its effect on neighbouring carbon
something, and as a scientist that’s
or hydrogens,’ says Ebel. ‘Being
obviously what you try and answer.’
able to detect heteroatom-specific
Detecting hydrogen bonds could
signatures directly would be one very
be just one of the potential future
strong point of AFM.’
applications of the STHM, Tautz
Tautz argues that it is STHM that
adds. ‘We now understand the basic
is more likely to become widely
mechanism, but there are still many
used, as it is much the simpler of
subtleties and interesting effects that Stefan Tautz’s STHM
the two techniques. ‘STHM is just
still need to be explored – and there
images of an aromatic
normal low temperature STM
may be some additional surprises
perylene derivative
still to come.’
showing possible H-bonds dosed with hydrogen. If you add
gaseous hydrogen into the sample
chamber then a thin layer of it
will automatically condense onto
the surface – so you always have
hydrogen at the junction between
the tip and the surface. We do AFM
as well as STM, so we know how
difficult it is to make those sensors.’
Champness for one is certain that
the techniques will become widely
adopted. ‘The images are so striking
that people will put the effort in to
try to do it. I’m sure other groups
are trying to do it now, and it’s only a
matter of time before it becomes
more widespread.’
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remains to be proven, he adds. ‘So far
it’s just a remarkable coincidence –
how it really works we don’t know
yet, that’s a direction of our
future work.’ The AFM
image of cephalandole
A captured by the IBM
team also appears
to show a hydrogen
bond, this time
intramolecular.
Champness, who
studies self-assembly of
molecules on surfaces,
thinks that these
imaging techniques
would be well suited
to answering some of
the difficult questions
faced by researchers
in his field. ‘There
can be questions
over orientation of
molecules, what atom is interacting
with what other atom, and
particularly where the hydrogen
atoms are, a question that can be very
difficult to answer.’
However, it is the possibility of
imaging hydrogen bonds that would
be particularly useful, Champness
adds. ‘In some sense, hydrogen bonds
are just another level of bonding
– but they are fundamental to life
on earth, and our understanding of
them is not complete. In our work we
rely on hydrogen bonds, but there are