From nanotechnology to nanomedicine: applications to cancer research.

Scientific advances have significantly improved the practice of medicine by providing objective and quantitative means for exploring the human body and disease states. These innovative technologies have already profoundly improved disease detection, imaging, treatment and patient follow-up. Today's analytical limits are at the nanoscale level (one-billionth of a meter) enabling a detailed exploration at the level of DNA, RNA, proteins and metabolites which are in fact nano-objects. This translational review aims at integrating some recent advances from micro- and nano-technologies with high potential for improving daily oncology practice.

Fluorescence relaxation in the near-field of a mesoscopic metallic particle: distance dependence and role of plasmon modes.

We analytically and numerically analyze the fluorescence decay rate of a quantum emitter placed in the vicinity of a spherical metallic particle of mesoscopic size (i.e with dimensions comparable to the emission wavelength). We discuss the efficiency of the radiative decay rate and non-radiative coupling to the particle as well as their distance dependence. The electromagnetic coupling mechanisms between the emitter and the particle are investigated by analyzing the role of the plasmon modes and their nature (dipole, multipole or interface mode). We demonstrate that near-field coupling can be expressed in a simple form verifying the optical theorem for each particle modes.

Probing physical properties of confined fluids within individual nanobubbles.

Spatially resolved electron energy-loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM) has been used to investigate a He fluidic phase in nanobubbles embedded in a metallic Pd(90)Pt(10) matrix. Using the 1s-->2p excitation of the He atoms, maps of the He density and pressure in bubbles of different diameters have been realized, to provide an indication of the bubble formation mechanism. Detailed local variations of the He K-line characteristics have been measured and interpreted as modifications of the electromagnetic properties of the He atom close to a metallic interface, which affects a correct estimation of the densities within the smallest bubbles.

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane.

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy.

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.

Micromechanical measurement of active sites on silicon nitride using surface free energy variation.

We have investigated chemical reactions between adsorbed water and active sites on a silicon nitride surface as a function of temperature and relative humidity using microcantilevers. Effects that might produce a change in the response of the microcantilever, such as a mass adsorption, surface tension of the adsorbed water, and changes in thermal conductivity, were systematically investigated. It is shown that the judicious choice of experimental conditions could make these effects essentially inconsequential in comparison with the instrument response produced by the change in free surface energy of the microcantilever due to the chemical reactions. Using this method, the variation in free surface energy when changing from dry to high humidity conditions was found and the number of active sites that reacted was estimated. This method may be extended to other problems for use in determining surface free energy change and thus the density of reactant sites under different conditions of interest.

Measuring magnetic susceptibilities of nanogram quantities of materials using microcantilevers.

We describe a novel technique for measuring magnetic susceptibilities of nanogram quantities of magnetic materials that utilizes the extreme force sensitivity of microcantilevers. The magnetic force acting on samples attached to the free end of a cantilever can be measured as changes in the resonance response of the cantilever. The shift in resonance frequency of the cantilever is proportional to the field gradient, whereas the deflection of a cantilever is proportional to the magnetic force. The magnetic susceptibility measurement is based on comparison of the forces acting on the sample and a reference material in the same magnetic field and field gradient. We have determined the magnetic susceptibilities of nanogram quantities of many paramagnetic materials. The measured magnetic susceptibilities show excellent agreement with values found in the literature.

Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal.

Difficulties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous buffer. A simple procedure that drastically enhances the successful imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a deflection signal, concomitant with the damping of the amplitude that can be followed by amplitude-distance curves. For intact cells, the evolution of the deflection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in finding the appropriate tapping conditions, the deflection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new field of applications for the atomic force microscope.

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.

Atomic force microscopy of renal cells: limits and prospects.

In this brief review, we present three-dimensional images of living Madin-Darby canine kidney (MDCK) cells and CV-1 cells that illustrate the possibilities and limits in the use of atomic force microscopy (AFM) for studying the topography of the cell surfaces and of isolated biological membranes. We show that microvilli can be imaged at the surface of living epithelial cells. However, when these microvilli are abundant and close to each other, the geometry of the AFM tip only allows an access to the upper part of the structures and precludes nanometer range imaging of the cell surface. Such a nanometer range imaging was obtained with other cell types like CV-1 cells and with isolated biological membranes. It reveals that protruding particles 5 to 60 nm xy size, likely corresponding to membranes proteins, occupy most of the membrane surface. These images indicate that the AFM already gives an access to the cell surface structure at the mesoscopic scale, which constitutes a major step for the understanding of the structure-function relationships in membranes. Perspectives for a further step, the imaging at molecular resolution of membranes, are discussed.

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy.

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to be a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.