The lateral organization of lipid components within membranes is usually investigated with fluorescence microscopy, which, though highly sensitive, introduces bulky fluorophores that might alter the behavior of the components they label. Secondary ion mass spectroscopy performed with a NanoSIMS 50 instrument also provides high lateral resolution and sensitivity, and many species can be observed in parallel without the use of bulky labels. A tightly focused beam (approximately 100 nm) of Cs ions is scanned across a sample, and up to five of the resulting small negative secondary ions can be simultaneously analyzed by a high-resolution mass spectrometer. Thin layers of (15)N- and (19)F-labeled proteins were microcontact-printed on an oxidized silicon substrate and imaged using the NanoSIMS 50, demonstrating the sensitivity and selectivity of this approach. Supported lipid bilayers were assembled on an oxidized silicon substrate, then flash-frozen and freeze-dried to preserve their lateral organization. Lipid bilayers were analyzed with the NanoSIMS 50, where the identity of each specific lipid was determined through detection of its unique secondary ions, including (12)C(1)H(-), (12)C(2)H(-), (13)C(-), (12)C(14)N(-), and (12)C(15)N(-). Steps toward obtaining quantitative composition analysis of lipid membranes that varied spatially in isotopic composition are presented. This approach has the potential to provide a composition-specific analysis of membrane organization that compliments other imaging modalities.
Interpretation of adsorption kinetics measured with a quartz crystal microbalance (QCM) can be difficult for adlayers undergoing modification of their mechanical properties. We have studied the behavior of the oscillation amplitude, A(0), and the decay time constant, tau, of quartz during adsorption of proteins and cells, by use of a home-made QCM. We are able to measure simultaneously the frequency, f, the dissipation factor, D, the maximum amplitude, A(0), and the transient decay time constant, tau, every 300 ms in liquid, gaseous, or vacuum environments. This analysis enables adsorption and modification of liquid/mass properties to be distinguished. Moreover the surface coverage and the stiffness of the adlayer can be estimated. These improvements promise to increase the appeal of QCM methodology for any applications measuring intimate contact of a dynamic material with a solid surface.
Cell attachment and spreading on solid surfaces was investigated with a home-made quartz crystal microbalance (QCM), which measures the frequency, the transient decay time constant and the maximal oscillation amplitude. Initial interactions of the adsorbing cells with the QCM mainly induced a decrease of the frequency, coincident with mass adsorption. After about 80 min, the frequency increased continuously and after several hours exceeded the initial frequency measured before cell adsorption. Phase contrast and fluorescence microscopy indicated that the cells were firmly attached to the quartz surface during the frequency increase. The measurements of the maximal oscillation amplitude and the transient decay time constant revealed changes of viscoelastic properties at the QCM surface. An important fraction of these changes was likely due to alterations of cytosolic viscosity, as suggested by treatments of the attached cells with agents affecting the actin and microtubule cytoskeleton. Our results show that viscosity variations of cells can affect the resonance frequency of QCM in the absence of apparent cell desorption. The simultaneous measurements of the maximal oscillation amplitude, the transient decay time constant and the resonance frequency allow an analysis of cell adsorption to solid substratum in real time and complement cell biological methods.
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