Uranium single atoms (circled) and small crystallites on a carbon support imaged simultaneously using a scanning probe to produce forward scattering through the sample (top) and backward scattering emerging from the surface (bottom). Center panel shows superimposition of the two in red (bulk) and green (surface). Atoms not seen in the lower image are on the bottom surface of the support. Source: Brookhaven National Laboratory

Scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory, in collaboration with researchers from Hitachi High Technologies Corp., have demonstrated a new scanning electron microscope capable of selectively imaging single atoms on the top surface of a specimen while a second, simultaneous imaging signal shows atoms throughout the sample’s depth. This new tool, located at Brookhaven Lab’s Center for Functional Nanomaterials (CFN), will greatly expand scientists’ ability to understand and ultimately control chemical reactions, such as those of catalysts in energy-conversion devices.
Like all scanning electron microscopes, the new tool probes a sample with an electron beam focused to a tiny spot and detects so-called secondary electrons emitted by the sample to reveal its surface structure and topography. Though this technique has been a workhorse of surface imaging in industrial and academic laboratories for decades, its resolution has left much to be desired because of imperfect focusing due to lens aberrations.
Using a newly developed spherical aberration corrector, the new tool corrects these distortions to create a smaller probe with significantly increased brightness.
The new device also employs specialized electron optics to channel the emitted secondary electrons to the detector. The result is a fourfold improvement in resolution to below one tenth of a nanometer — and thus, the ability to image single atoms.
Additional detectors, located below the sample, detect electrons transmitted through the sample, revealing details about the entire structure at the exact instant the “shutter” snapped to record each pixel of the surface image. This simultaneous imaging allows the scientists to correlate information in the two images to understand precisely what is happening on the surface and throughout the sample at the same time.
Because of its extreme sensitivity, the new microscope must be kept isolated from a range of environmental effects such as variations in temperature, mechanical vibrations, and electromagnetic fields. Even the slightest waft of air could cause distortions in the images.

Original publication:
Zhu Y., Inada H., Nakamura K., Wall J.: Imaging single atoms using secondary electrons with an aberration-corrected microscope. Nat Mater. 2009 Sep 20. [Epub ahead of print]

http://www.bnl.gov

Dr Berenguer is part of Prof Ian Robinson’s group in the London Centre for Nanotechnology. This group is developing methods of using the coherence properties of these X-rays for imaging materials on the nanoscale. They use new synchrotron X-ray sources with extremely high brightness such as the Diamond Light Source on the Harwell campus near Oxford. While new light lines at the Diamond Light Source are still under construction, the London Centre Nanotechnology operates one of the experimental out-stations of the Advanced Photon Source (APS), an X-ray synchrotron in Chicago, USA. The group is focusing its efforts on X-rays because this type of light has small wavelengths and is strongly penetrating into material. There is thus an opportunity for imaging physical structures in three dimensions with resolution well beyond that of the visible light microscope. The group is also developing phase-contrast methods that are sensitive to nanoscale strains, or the detailed packing arrangement of molecules in biological tissues.

Original publication:
Berenguer de la Cuesta F, Wenger MP, Bean RJ, Bozec L, Horton MA, Robinson IK. : Coherent X-ray diffraction from collagenous soft tissues. Proc Natl Acad Sci U S A. 2009 Aug 24. [Epub ahead of print]

http://www.london-nano.com

Diffraction pattern of collagen obtain by Dr Berenguer and al during the scope of this research. Source: London Centre for Nanotechnology

Electron tomography is carried out at the nanoscale on individual cells. Conventionally, high-resolution imaging of biological specimens has been accomplished by cutting cells into thin sections (300 nanometers or less) and imaging each section separately. Although reconstructing an entire structure from thin sections is laborious, thin sections are used because images of thicker sections typically are blurred. Serial BF STEM tomography accomplishes the same work using fewer yet thicker specimen sections, leading to faster reconstruction of intact organelles, intracellular pathogens, and even entire mammalian cells.

Drs. Alioscka Sousa, Martin Hohmann-Marriott, Richard Leapman and colleagues in NIBIB, in collaboration with Dr. Joshua Zimmerberg and colleagues in the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), demonstrated feasibility and advantages of BF-STEM tomography in a study of red blood cells infected with Plasmodium falciparum, a parasite that causes malaria. High-resolution 3D reconstructions of entire cells were generated by serially imaging just a few thick sections. The intricate system of red blood cell and parasite membranes, as well as several organelles, can be seen in detail.

Original publication:
Hohmann-Marriott MF, Sousa AA, Azari AA, Glushakova S, Zhang G, Zimmerberg J & Leapman RD.: Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nature Methods, Published online: 30 August 2009 | doi:10.1038/nmeth.1367.

http://www.nih.gov

Christine Baumgart now discovered what exactly is measured by Kelvin probe force microscopy. It is the electric potential which is needed to move electrons or holes from the inside to the surface of a semiconductor. Her new findings will simplify the microscopic technique itself, and will lead to unambiguous and reproducible results concerning the structure and electronic properties of samples. Also, Kelvin probe force microscopy, which has been used mainly in materials science and semiconductor physics so far, is likely to become more attractive for other areas like biotechnology.

But how exactly does a Kelvin probe force microscope work? The tip is deflected by the electrostatic force between cantilever and sample when moved over the sample. By applying bias to the sample, electrons and holes are moved to the surface of the semiconductor and the electrostatic force decreases. The cantilever moves back to its original position and the applied bias is stored as the signal measured. To be more precise, there is a quantitative relation between the measured Kelvin bias and the difference between the calculated Fermi energy and respective semiconductor band edge independent of the work function of the probing microscope tip. Thus, Christine Baumgart’s novel explanation of how the Kelvin probe force microscope works elucidates why the signal depends on the bias necessary for injecting majority charge carriers towards the interface between insulator and semiconductor.

Original publication:

Baumgart C., Helm M., Schmidt H., (2009): Quantitativ dopant profiling in semiconducters: A Kelvin probe force microscopy model. DOI: 10.1103/PhysRevB.80.085305.

https://www.fzd.de

Schematic drawing of a Kelvin probe force microscopy probe above a doped semiconductor with a thin oxide layer (grey blue atomic layer). Occupied surface states at the interface between the oxide layer and the semiconductor are animated in red and the same number of unscreened dopant atoms is animated in dark blue. Left: The resulting asymmetric electric dipole causes the deflection of the probe. Center: By applying a bias mobile majority charge carriers are injected into the semiconductor (animated in orange) and screen the unscreened ionized dopant atoms. Right: As a result the electrostatic force onto the cantilever vanishes. The cantilever moves back to its normal position. The applied bias is measured and depends on the concentration of dopant atoms. Picture: Sander Münster, Kunstkosmos

The results push the exploration of using molecules and atoms at the smallest scale and could greatly impact the field of nanotechnology, which seeks to understand and control some of the smallest objects know to mankind.

As reported in the August 28 issue of Science magazine, IBM Research – Zurich scientists Leo Gross, Fabian Mohn, Nikolaj Moll and Gerhard Meyer, in collaboration with Peter Liljeroth of Utrecht University, used an AFM operated in an ultrahigh vacuum and at very low temperatures (–268°C or – 451°F) to image the chemical structure of individual pentacene molecules. With their AFM, the IBM scientists, for the first time ever, were able to look through the electron cloud and see the atomic backbone of an individual molecule. While not a direct technological comparison, this is reminiscent of X-rays that pass through soft tissue to enable clear images of bones.

Original publication:
Gross L, Mohn F, Moll N, Liljeroth P, and Meyer G, (2009): The Chemical Structure of a Molecule Resolved by Atomic Force Microscopy, Science, Vol. 325, Issue 5944, pp. 1110 – 1114 (28 August 2009).

http://www.zurich.ibm.com

The delicate inner structure of a pentacene molecule imaged with an atomic force microscope. For the first time, scientists achieved a resolution that revealed the chemical structure of a molecule. The hexagonal shapes of the five carbon rings in the pentacene molecule are clearly resolved. Even the positions of the hydrogen atoms around the carbon rings can be deduced from the image. (Pixels correspond to actual data points). Source: IBM Research – Zurich

Two-photon-microscopy of neuron. Source: FMI

Fifty years after it was originally discovered, scientists at the Friedrich Miescher Institute for Biomedical Research have elucidated the function of a microscopic network of tubules found in neurons. This structure modulates the strength of connections between two neurons, thereby contributing to our ability to learn and to adapt to new situations.

In the current online issue of the Proceedings of the National Academy of Sciences, Oertner’s group describes how the microscopic network of tubules known as the endoplasmic reticulum (ER) modulates the strength of connections between neurons. Neural connections play an important role in our ability to learn new information and constantly adapt to new conditions. In the brain, synaptic connections between neurons are thus continually formed, strengthened or weakened.

In addition, there are connections that remain stable – for example, when we store an important memory for many years. Connections of both types coexist, and their close proximity was one of the first key findings of Thomas Oertner’s study: the two types occur side by side on the same neuron and are individually controlled.

Observations and comparisons of individual dendritic spines and synapses are anything but routine. The findings just published were only possible thanks to a new method known as two photon microscopy. In this imaging technique, a pulsed infrared laser is used to excite fluorescence in a dye molecule in a cell. It is a gentle method of investigating cells, which yields unique, high-resolution images. Thomas Oertner’s research group is one of only a few worldwide that use this technique to optically stimulate and observe individual synapses, and to measure their activity. As equipment of this type cannot simply be purchased off the shelf, these innovative microscopes are custom-built and refined by Thomas Oertner himself. 

Original publication:
Holbro N et al. (2009) Differential distribution of endoplasmic reticulum controls metabotropic signaling and plasticity at hippocampal synapses. PNAS, 18 August 2009, doi: 10.1073/pnas.0905110106 -> Online publication

http://www.fmi.ch

Kelvin Probe Force Microscopy schematic

Surface charges play a key role in determining the structure and function of proteins, DNA and larger biomolecular structures. For example, negatively charged DNA strands electrostatically interact with histone proteins, transcription factors, or polymerases thereby influencing the read-out of genetic information and the development of cancer. Similarly, the central process of protein folding and protein interaction, often governed by charges, is the major factor in protein-folding diseases such as Alzheimer’s or Parkinson’s Disease. However, thus far there have been no experimental methods to spatially resolve the electrostatic surface potential of individual biological molecules. In general, the investigation of individual molecules can shed light on their dynamic behaviour or on static heterogeneity which is masked in ensemble measurements.

A collaborative effort between researchers from the London Centre for Nanotechnology (Bart W Hoogenboom), King’s College London (Carl Leung, Patrick Mesquida) and UCL Chemistry (Stefan Howorka, Helen Kinns) has led to the first measurements of the electrostatic surface potential of individual DNA and avidin molecules with nanometre resolution using Kelvin Probe Force Microscopy (KPFM) in air.

Kelvin Probe Force Microscopy (KPFM) can measure surface charges by contactless recording of the electrostatic force between a conductive Atomic Force Microscope tip and a biomolecule on a support. To achieve this, the AFM tip is simultaneously excited at its mechanical resonance frequency and by an electrical (AC) voltage. This periodic electrical voltage on the tip leads to a force between the tip and the charges on the biomolecule, which is recorded by means of a lock-in amplifier and nullified by the Kelvin mode feedback by applying a separate DC voltage (not shown). The polarity and magnitude of this DC voltage corresponds to the local surface charge profile (in mV) which is recorded simultaneously with the topography of the biomolecule.

The investigation led at the London Centre for Nanotechnology also show, for the first time, the surface potential of buffer salts shielding DNA molecules on a surface, which would not be possible with conventional ensemble techniques. It is anticipated that the ability to visualize the electrostatic surface potentials of individual proteins and DNA at molecular resolution will be an important tool in fundamental biophysical research and in the fields of biosensing and bio-nanoelectronics.

Original Publication:

Leung C, Kinns H, Hoogenboom BW, Howorka S, Mesquida P. (2009): Imaging surface charges of individual biomolecules.  Nano Lett. 2009 Jul;9(7):2769-73.

 http://www.london-nano.com

The researchers demonstrated their new understanding of how the instrument – the atomic force microscope – works in water to show detailed properties of a bacterial membrane and a virus called Phi29, said Arvind Raman, a Purdue professor of mechanical engineering. An atomic force microscope uses a tiny vibrating probe to yield information about materials and surfaces on the scale of nanometers, or billionths of a meter. Because the instrument enables scientists to “see” objects far smaller than possible using light microscopes, it could be ideal for studying molecules, cell membranes and other biological structures. The best way to study such structures is in their wet, natural environments. However, the researchers have now discovered that in some respects the vibrating probe’s tip behaves the opposite in water as it does in air, said Purdue mechanical engineering doctoral student John Melcher. The probe is caused to oscillate by a vibrating source at its base. However, the tip of the probe oscillates slightly out of synch with the oscillations at the base. This difference in oscillation is referred to as a “phase contrast,” and the tip is said to be out of phase with the base.

Although these differences in phase contrast reveal information about the composition of the material being studied, data can’t be properly interpreted unless researchers understand precisely how the phase changes in water as well as in air, Raman said.

If the instrument is operating in air, the tip’s phase lags slightly when interacting with a viscous material and advances slightly when scanning over a hard surface. Now researchers have learned the tip operates in the opposite manner when used in water: it lags while passing over a hard object and advances when scanning the gelatinous surface of a biological membrane.

Researchers deposited the membrane and viruses on a sheet of mica. Tests showed the differing properties of the inner and outer sides of the membrane and details about the latticelike protein structure of the membrane. Findings also showed the different properties of the balloonlike head, stiff collar and hollow tail of the Phi29 virus, called a bacteriophage because it infects bacteria.

Original Publication:

Melcher J, Carrasco C, Xu X, Carrascosa JL, Gómez-Herrero J, José de Pablo P, Raman A. (2009): Origins of phase contrast in the atomic force microscope in liquids. Proc Natl Acad Sci U S A. 2009 Aug 18;106(33):13655-60. Epub 2009 Aug 5.

Researchers in the United States and Spain have discovered that an atomic force microscope - a tool widely used in nanoscale imaging - works differently in watery environments, a step toward better using the instrument to study biological molecules and structures. The researchers demonstrated their new understanding of how the instrument works in water to show details of the mechanical properties of a virus called Phi29. The images in "a" and "c" show the topography, and the image in "b" shows the different stiffness properties of the balloonlike head, stiff collar and hollow tail of the Phi29 virus, called a bacteriophage because it infects bacteria. (C. Carrasco-Pulido, P. J. de Pablo, J. Gomez-Herrero, Universidad Autonoma de Madrid, Spain)

http://news.uns.purdue.edu

A new technique known as “liquid cell in situ transmission electron microscopy,” in which the powerful resolution capabilities of a transmission electron microscope (TEM) are brought to bear on a liquid cell that allows liquids to be observed inside a vacuum, enables the visualization of single nanoparticles in solution. The Berkeley researchers deployed this technique on NCEM’s JEOL 3010 In-Situ microscope. Utilizing an electron beam operating at 300 kilovolts of energy, the JEOL 3010 provides outstanding specimen penetration and spatial resolution of about 8 angstroms through the thick liquid cell sample.

Original publication:

Zheng H, Smith RK, Jun YW, Kisielowski C, Dahmen U, Alivisatos AP (2009): Observation of Single Colloidal Platinum Nanocrystal Growth Trajectories. Science Jun 5;324(5932):1309-12.

http://newscenter.lbl.gov

The research team led by Dr. Timm Schroeder, stem cell researcher at Helmholtz Center Munich, Germany has developed a new bioimaging method for observing the differentiation of hematopoietic progenitor cells (HPC) at the single-cell level. With this method the researchers were able to prove for the first time that not only cell-intrinsic mechanisms, but also external environmental factors such as growth factors can control HPC lineage choice directly. The findings, published in Science, provide an essential building block for understanding the molecular mechanisms of hematopoiesis and are an important prerequisite for optimizing therapeutic stem cell applications.

With the new bioimaging techniques developed by Dr. Schroeder’s team, progenitor cells could be observed for a longer period and on the single-cell level. Depending on the kind of cytokines present, after a few days the HPC cultures contained only one cell type. The question remained unanswered whether this was a consequence of direct cytokine regulation or merely the result of sorting out “erroneously differentiated” cells by cell death. Using the new bioimaging techniques for continuous single-cell observation, Dr. Michael Rieger and students in Dr. Schroeder’s research group showed for the first time that no cell death could be detected during the entire cell differentiation process. This proves unambiguously that HPC lineage choices can be steered by external environmental factors such as in this case by cytokines. The hematopoietic progenitor cells are “instructed” by cytokines.
www.helmholtz-muenchen.de

Original publication:
Rieger MA, Hoppe PS, Smejkal BM, Eitelhuber AC & Schroeder T (2009): Hematopoietic cytokines can instruct lineage choice. Science 325:217-218