Spatially resolved force spectroscopy of biological surfaces using the atomic force microscope.

The spatial distribution of intermolecular forces governs macromolecular interactions. The atomic force microscope, a relatively new tool for investigating interaction forces between nanometer-scale objects, can be used to produce spatially resolved maps of the surface or material properties of a sample; these include charge density, adhesion and stiffness, as well as the force required to break specific ligand-receptor bonds. Maps such as these will provide fundamental insights into biological structure and will become an important tool for characterizing technologically important biological systems.

Relative surface charge density mapping with the atomic force microscope.

An experimental approach for producing relative charge density maps of biological surfaces using the atomic force microscope is presented. This approach, called D minus D (D-D) mapping, uses isoforce surfaces collected at different salt concentrations to remove topography and isolate electrostatic contributions to the tip-sample interaction force. This approach is quantitative for surface potentials below 25 mV, and does not require prior knowledge of the cantilever spring constant, tip radius, or tip charge. In addition, D-D mapping does not require tip-sample contact. The performance of D-D mapping is demonstrated on surfaces of constant charge and varying topography (mechanically roughened mica and stacked bilayers of dipalmitolphosphatidylserine), a surface of varying charge and varying topography (patches of dipalmitolphosphatidylcholine on mica), and bacteriorhopsin membranes adsorbed to mica.

High-energy electron irradiation of proteins and nucleic acids: collisional stopping power and average energy loss.

Inactivation of proteins due to the direct action of ionizing radiation and the electron energy loss spectra of organic materials indicate that an average of 60-66 eV of energy is lost from high energy electrons in each inelastic collision with target molecules. The average energy loss per inelastic collision with high energy electrons in solid, carbon-based materials, proteins and nucleic acids is calculated from mass collisional stopping powers and empirical total inelastic cross-sections. Bragg's Additivity Law is used for the calculation of the mean excitation energy of molecules. For simple organic compounds, the calculated average energy loss is close to that obtained by direct observation of the energy loss suffered by electrons as they pass through thin films of organic material. The density effect correction for the rate of energy loss, important in the more complex case of proteins irradiated with 10 MeV electrons, is determined using the comparable mass collisional stopping power of water and proteins. In this manner, a value is obtained for the average energy per inelastic collision of high energy electrons with proteins, which is similar to the average energy per inactivating event of proteins. Analogous calculations for nucleic acids are also presented.

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.