Single-molecule super-resolution imaging in bacteria.

Bacteria have evolved complex, multi-component cellular machineries to carry out fundamental cellular processes such as cell division/separation, locomotion, protein secretion, DNA transcription/replication, or conjugation/competence. Diffraction of light has so far restricted the use of conventional fluorescence microscopy to reveal the composition, internal architecture and dynamics of these important machineries. This review describes some of the more recent advances on single-molecule super-resolution microscopy methods applied to bacteria and highlights their application to chemotaxis, cell division, DNA segregation, and DNA transcription machineries. Finally, we discuss some of the lessons learned from this approach, and future perspectives.

Salt concentration effects on equilibrium melting curves from DNA microarrays.

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.

Influence of an electric field on oriented films of DMPC/gramicidin bilayers: a circular dichroism study.

A film of oriented bilayers containing a mixture of gramicidin and dimyristoylphosphatidylcholine (DMPC) has been deposited on a fused-silica window coated with a 10 nm thick gold layer. The thin layer of gold allows the application of an electric potential across the film and the study of its influence on the structure and integrity of the bilayers. Electrochemical measurements, ellipsometry, and far-UV circular dichroism (CD) were employed to characterize the properties of the film of bilayers as a function of the potential applied to the gold electrode. For potentials across the film that are within the range approximately +300 to -150 mV the oriented film of bilayers is stable, and no change in the CD spectra of gramicidin molecule is observed. At more negative potentials, an increase in the film thickness and water content measured by ellipsometry indicated that the film swells and incorporates water, which causes a change in the circular dichroism spectrum of gramicidin molecules in the film. This transformation was interpreted as a change in the average orientation of gramicidin molecules within the film due to a decrease in the ordering of the molecules upon swelling.

SPR-imaging based assays on an oligonucleotide-array to analyze DNA lesions recognition and excision by repair proteins.

An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.

Point mutation detection by surface plasmon resonance imaging coupled with a temperature scan method in a model system.

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNA's melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations.

Temperature effects on DNA chip experiments from surface plasmon resonance imaging: isotherms and melting curves.

We present an analysis of hybridization experiments on a DNA chip studied by surface plasmon resonance imaging. The reaction constants at various temperatures and for different probe lengths are obtained from Langmuir isotherms and hybridization kinetics. The melting curves from temperature scans are also obtained without any labeling of the targets. The effects of the probe length on the hybridization thermodynamics, deduced from the temperature dependence of the reaction constants as well as from the melting curves, suggest dispersion in the length of the hybridization segments of the probes accessible to the targets. Those are, however, sufficient to suggest efficient point mutation detection from temperature scans.