Automated DNA Fragments Recognition and Sizing through AFM Image Processing

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes and for different background noises. The automated approach minimizes processing time with respect to manual and semi-automated DNA sizing. Moreover, the DNA molecule profile recognition can be used to perform further structural analysis. For computer-generated images, the root mean square error incurred by the automated algorithm in the length estimation is 0.6% for a 7.8 nm image pixel size and 0.34% for a 3.9 nm image pixel size. For AFM real images we obtain a distribution of lengths with a standard deviation of 2.3% of mean and a measured average length very close to the real one, with an error around 0.33%

New Light on Molecular and Materials Complexity: 4D Electron Imaging

In this Perspective, 4D electron imaging is highlighted, after introducing some concepts, with an overview of selected applications that span chemical reactions, molecular interfaces, phase transitions, and nano(micro)mechanical systems. With the added dimension of time in microscopy, diffraction, and electron-energy-loss spectroscopy, the focus is on direct visualization of structural dynamics with atomic and nanoscale resolution in the four dimensions of space and time. This contribution provides an expose of emerging developments and an outlook on future applications in materials and biological sciences

Electrophoretic separation of large DNAs using steric confinement

We report an alternative method for electrophoretic separation of large DNAs using steric confinement between solid walls, without gel or obstacles. The change of electrophoretic mobility vs confinement thickness is investigated using fluorescence video microscopy. We observe separation at small confinement thicknesses followed by a transition to the bulk behaviour (no separation) at a thickness of about 4 μm (a few radii of gyration for the studied DNA chains). We present tentative explanations of our original observations.Comment: to appear in JCI

Microbiology and atmospheric processes: Biological, physical and chemical characterization of aerosol particles

The interest in bioaerosols has traditionally been linked to health hazards for humans, animals and plants. However, several components of bioaerosols exhibit physical properties of great significance for cloud processes, such as ice nucleation and cloud condensation. To gain a better understanding of their influence on climate, it is therefore important to determine the composition, concentration, seasonal fluctuation, regional diversity and evolution of bioaerosols. In this paper, we will review briefly the existing techniques for detection, quantification, physical and chemical analysis of biological particles, attempting to bridge physical, chemical and biological methods for analysis of biological particles and integrate them with aerosol sampling techniques. We will also explore some emerging spectroscopy techniques for bulk and single-particle analysis that have potential for in-situ physical and chemical analysis. Lastly, we will outline open questions and further desired capabilities (e. g., in-situ, sensitive, both broad and selective, on-line, time-resolved, rapid, versatile, cost-effective techniques) required prior to comprehensive understanding of chemical and physical characterization of bioaerosols

Microscopic Analysis of DNA and DNA-Protein Assembly by Transmission Electron Microscopy, Scanning Tunneling Microscopy and Scanning Force Microscopy

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA super-coiling were analyzed using electron microscopy. In parallel, this Mg2+/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or in liquid by scanning force microscopy. An advantage of such a correlative approach is that parallel imaging can reveal complementary information. The benefit of such a combined approach in analysis of protein-induced DNA bending is discussed

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein–DNA Complexes That Carry Out DNA Repair

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein–protein and protein–DNA interactions. It can be used to capture snapshots of protein–DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein–protein and protein–DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein–protein and protein–DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein–DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein–protein and protein–nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure–function relationships that govern DNA repair processes

Nano-imaging and its applications to biomedicine

Nanotechnology tools, such as Atomic Force Microscopy (AFM), are now becoming widely used in life sciences and biomedicine. AFM is a versatile technique that allows studying at the nanoscale the morphological, dynamic, and mechanical properties of biological samples, such as living cells, biomolecules, and tissues under physiological conditions. In this article, an overview of the principles of AFM will be first presented and this will be followed by discussion of some of our own recent work on the applications of AFM imaging to biomedicine