Imaging Collagen with X-rays

September 21, 2009

Coherent X-ray Diffraction patterns of collagen in soft tissues have been measured for the first time by Dr Felisa Berenguer (London Centre for Nanotechnology) with her colleagues. This development opens doors to better understanding of living tissues like skin and bones, as well as the bio-mineralization processes which turn flexible collagen into semi-flexible cartilage and eventually into rigid bones. In a distant future, the understanding of the collagen structure will eventually lead to cures for of bone diseases, notably osteoporosis, or assist ongoing efforts to develop artificial skin.

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]

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

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

Imaging Surface Charges on Individual Biomolecules

September 2, 2009
Kelvin Probe Force Microscopy schematic

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.