Probing nanomechanical properties from biomolecules to living cells

Atomic force microscopy is being increasingly used to explore the physical properties of biological structures. This technique involves the application of a force to the sample and a monitoring of the ensuing deformation process. The available experimental setups can be broadly divided into two categories, one of which involves a stretching and the other an indentation of the organic materials. In this review, we will focus on the indentation technique and will illustrate its application to biological materials with examples that range from single molecules to living cell

Elastic properties of grafted microtubules

We use single-particle tracking to study the elastic properties of single microtubules grafted to a substrate. Thermal fluctuations of the free microtubule's end are recorded, in order to measure position distribution functions from which we calculate the persistence length of microtubules with contour lengths between 2.6 and 48 micrometers. We find the persistence length to vary by more than a factor of 20 over the total range of contour lengths. Our results support the hypothesis that shearing between protofilaments contributes significantly to the mechanics of microtubules.Comment: 9 pages, 3 figure

Nanomechanical sensor applied to blood culture pellets: a fast approach to determine the antibiotic susceptibility against agents of bloodstream infections.

The management of bloodstream infection, a life-threatening disease, largely relies on early detection of infecting microorganisms and accurate determination of their antibiotic susceptibility to reduce both mortality and morbidity. Recently we developed a new technique based on atomic force microscopy capable of detecting movements of biologic samples at the nanoscale. Such sensor is able to monitor the response of bacteria to antibiotic's pressure, allowing a fast and versatile susceptibility test. Furthermore, rapid preparation of a bacterial pellet from a positive blood culture can improve downstream characterization of the recovered pathogen as a result of the increased bacterial concentration obtained. Using artificially inoculated blood cultures, we combined these two innovative procedures and validated them in double-blind experiments to determine the susceptibility and resistance of Escherichia coli strains (ATCC 25933 as susceptible and a characterized clinical isolate as resistant strain) towards a selection of antibiotics commonly used in clinical settings. On the basis of the variance of the sensor movements, we were able to positively discriminate the resistant from the susceptible E. coli strains in 16 of 17 blindly investigated cases. Furthermore, we defined a variance change threshold of 60% that discriminates susceptible from resistant strains. By combining the nanomotion sensor with the rapid preparation method of blood culture pellets, we obtained an innovative, rapid and relatively accurate method for antibiotic susceptibility test directly from positive blood culture bottles, without the need for bacterial subculture

The Dynamics of Single-Cell Nanomotion Behaviour of Saccharomyces cerevisiae in a Microfluidic Chip for Rapid Antifungal Susceptibility Testing

The fast emergence of multi-resistant pathogenic yeasts is caused by the extensive-and sometimes unnecessary-use of broad-spectrum antimicrobial drugs. To rationalise the use of broad-spectrum antifungals, it is essential to have a rapid and sensitive system to identify the most appropriate drug. Here, we developed a microfluidic chip to apply the recently developed optical nanomotion detection (ONMD) method as a rapid antifungal susceptibility test. The microfluidic chip contains no-flow yeast imaging chambers in which the growth medium can be replaced by an antifungal solution without disturbing the nanomotion of the cells in the imaging chamber. This allows for recording the cellular nanomotion of the same cells at regular time intervals of a few minutes before and throughout the treatment with an antifungal. Hence, the real-time response of individual cells to a killing compound can be quantified. In this way, this killing rate provides a new measure to rapidly assess the susceptibility of a specific antifungal. It also permits the determination of the ratio of antifungal resistant versus sensitive cells in a population

Probing nanomechanical properties from biomolecules to living cells

Atomic force microscopy is being increasingly used to explore the physical properties of biological structures. This technique involves the application of a force to the sample and a monitoring of the ensuing deformation process. The available experimental setups can be broadly divided into two categories, one of which involves a stretching and the other an indentation of the organic materials. In this review, we will focus on the indentation technique and will illustrate its application to biological materials with examples that range from single molecules to living cells

Atomic Force Microscopy Imaging of Living Cells

Over the last two decades, Atomic Force Microscopy (AFM) has emerged as the tool of choice to image living organisms in a near-physiological environment. Whereas fluorescence microscopy techniques allow labeling and tracking of components inside cells and the observation of dynamic processes, AFM is mainly a surface technique that can be operated on a wide range of substrates including biological samples. AFM enables extraction of topographical, mechanical and chemical information from these sample

Discrete reduction patterns of parvalbumin and calbindin D-28k immunoreactivity in the dorsal lateral geniculate nucleus and the striate cortex of adult macaque monkeys after monocular enucleation

We analyzed the immunohistochemical distribution of the two calcium-binding proteins, parvalbumin (PV) and calbindin D-28k (CB), in the primary visual cortex and lateral dorsal geniculate nucleus (dLGN) of monocularly enucleated macaque monkeys (Macaca fascicularis and Macaca nemestrind) in order to determine how the expression of PV and CB is affected by functional inactivity. The monkeys survived 1-17 weeks after monocular enucleation. The distribution pattern of each of the proteins was examined immunocytochemically using monoclonal antibodies and compared with that of the metabolic marker cytochrome oxidase (CO). We recorded manually the number of immunostained neurons and estimated the concentration of immunoreactive staining product using a computerized image-acquisition system. Our results indicate a decrease of approximately 30% in the labeling of PV-immunoreactive (ir) neuropil particularly in those layers of denervated ocular-dominance columns receiving the geniculocortical input. There was no change in the number of PV-ir neurons in any compartment irrespective of the enucleation interval. For CB-ir, we found a 20% decrease in the neuropil labeling in layer 2/3 of the denervated ocular-dominance columns. In addition, a subset of pyramidal CB-ir neurons in layers 2 and 4B, which are weakly stained in control animals, showed decreased labeling. In the dLGN of enucleated animals, PV-ir and CB-ir were decreased only in the neuropil of the denervated layers. From these results, we conclude that cortical interneurons and geniculate projection neurons still express PV and CB in their cell bodies after disruption of the direct functional input from one eye. The only distinct decrease of PV and CB expression is seen in axon terminals from retinal ganglion cells in the dLGN, and in the axons and terminals of both geniculocortical projection cells and cortical interneurons in the cerebral corte

Probing nanomechanical properties from biomolecules to living cells.

Atomic force microscopy is being increasingly used to explore the physical properties of biological structures. This technique involves the application of a force to the sample and a monitoring of the ensuing deformation process. The available experimental setups can be broadly divided into two categories, one of which involves a stretching and the other an indentation of the organic materials. In this review, we will focus on the indentation technique and will illustrate its application to biological materials with examples that range from single molecules to living cells