A new frequency modulation (FM) technique has been demonstrated which ennances the sensitivity of attractive mode force microscopy by an order of magnitude or more. Increased sensitivity is made possible by operating in a moderate vacuum ( < 10 -' Torr), which increases the Q of the vibrating cantilever. In the FM technique, the cantilever serves as the frequency determining element of an oscillator. Force gradients acting on the cantilever cause instantaneous frequency modulation of the oscillator output, which is demodulated with a FM detector. Unlike conventional "slope detection," the FM technique offers increased sensitivity through increased Q without restricting system bandwidth. Experimental comparisons of FM detection in vacuum (Q-50 000) versus slope detection in air (Q-100) demonstrated an improvement of more than 10 times in sensitivity for a fixed bandwidth. This improvement is evident in images of magnetic transitions on a thin-film CoPtCr magnetic disk. In the future, the increased sensitivity offered by this technique should extend the range of problems accessible by force microscopy.

A mechanical degenerate parametric amplifier has been devised which greatly increases the motional response of a microcantilever for small harmonic force excitations. The amplifier can improve force detection sensitivity for measurements dominated by sensor noise or backaction effects and can also produce mechanical squeezed states. In an initial squeezing demonstration, the thermal noise (Brownian motion) of the cantilever was reduced in one phase by 4.9 dB

Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation. INTRODUCTIONMutations affecting mitochondrial oxidative phosphorylation cause a variety of multisystem disorders (DiMauro and Schon, 2003;Taylor and Turnbull, 2005) with an estimated incidence of 1:5000 live births (Thorburn, 2004). The majority of these mutations are found in the mitochondrial genome (mtDNA), which encodes the core hydrophobic proteins involved in oxidative phosphorylation and some of the molecules required for their expression, such as tRNAs and rRNAs. The mutant mtDNA genomes generally coexist alongside wild-type copies in affected patients, a situation referred to as heteroplasmy. The segregation pattern of heteroplasmic DNAs in different tissues is an important determinant of the severity of the clinical phenotype. To develop an animal model of heteroplasmy, our laboratory generated mice containing polymorphisms in otherwise normal mtDNA, and we identified three nuclear quantitative trait loci that affect mtDNA segregation in liver, spleen, and kidney (Jenuth et al., 1997;Battersby et al., 2003). The selection of one variant of the mtDNA over another could be explained by differences in their organization or packaging.Mammalian mtDNA is packaged in protein-DNA complexes termed nucleoids, which can be visualized as small submitochondrial bodies in the matrix (Nass, 1969). The nucleoid contains two to seven genomes, depending on cell type, and it is present as 450 -800 distinct foci in cultured cells (Nass, 1969;Iborra et al., 2004;Legros et al., 2004). Several mammalian mitochondrial nucleoid proteins have been identified after enrichment by sedimentation and immunoprecipitation (Wang and Bogenhagen, 2006;He et al., 2007). However, other than the components of the mitochondrial replisome and transcription apparatus, the only proteins that have been fo...

Uptake and intracellular trafficking of hydrogel nanoparticles (NPs) of N,N-diethyl acrylamide and 2-hydroxyethyl methacrylate crosslinked with N,N′-methylene-bis-acrylamide were studied with a RAW 264.7 murine macrophage cell line. Results show that the uptake rate, the mechanism of internalization and the concentration of internalized NPs are correlated to the NP Young modulus. Soft NPs are found to be internalized preferentially via macropinocytosis while the uptake of stiff NPs is mediated by a clathrin-dependent mechanism. NPs with an intermediate Young modulus exhibit multiple uptake mechanisms. The accumulation rate of the NPs into lysosomal compartments of the cell is also dependent on the NP elasticity. Our results suggest that control over the mechanical properties of hydrogel NPs can be used to tailor the cellular uptake mechanism and kinetics of drug delivery

Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100 Hz. The resulting elastic storage modulus (G') and dissipative modulus (G'') increased dramatically, with hysteresivity (eta = G''/G') showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

The surface stress induced during the formation of alkanethiol self-assembled monolayers (SAMs) on gold from the vapor phase was measured using a micromechanical cantilever-based chemical sensor. Simultaneous in situ thickness measurements were carried out using ellipsometry. Ex situ scanning tunneling microscopy was performed in air to ascertain the final monolayer structure. The evolution of the surface stress induced during coverage-dependent structural phase transitions reveals features not apparent in average ellipsometric thickness measurements. These results show that both the kinetics of SAM formation and the resulting SAM structure are strongly influenced both by the surface structure of the underlying gold substrate and by the impingement rate of the alkanethiol onto the gold surface. In particular, the adsorption onto gold surfaces having large, flat grains produces high-quality self-assembled monolayers. An induced compressive surface stress of 15.9 ± 0.6 N/m results when a c(4×2) dodecanethiol SAM forms on gold. However, the SAMs formed on small-grained gold are incomplete and an induced surface stress of only 0.51 ± 0.02 N/m results. The progression to a fully formed SAM whose alkyl chains adopt a vertical (standing-up) orientation is clearly inhibited in the case of a small-grained gold substrate and is promoted in the case of a large-grained gold substrate.

Many interactions drive the adsorption of molecules on surfaces, all of which can result in a measurable change in surface stress. This article compares the contributions of various possible interactions to the overall induced surface stress for cantilever-based sensing applications. The surface stress resulting from adsorption-induced changes in the electronic density of the underlying surface is up to 2-4 orders of magnitude larger than that resulting from intermolecular electrostatic or Lennard-Jones interactions. We reveal that the surface stress associated with the formation of high quality alkanethiol self-assembled monolayers on gold surfaces is independent of the molecular chain length, supporting our theoretical findings. This provides a foundation for the development of new strategies for increasing the sensitivity of cantilever-based sensors for various applications.

Single-electron charging in an individual InAs quantum dot was observed by electrostatic force measurements with an atomic-force microscope (AFM). The resonant frequency shift and the dissipated energy of an oscillating AFM cantilever were measured as a function of the tip-back electrode voltage, and the resulting spectra show distinct jumps when the tip was positioned above the dot. The observed jumps in the frequency shift, with corresponding peaks in dissipation, are attributed to a single-electron tunneling between the dot and the back electrode governed by the Coulomb blockade effect, and are consistent with a model based on the free energy of the system. The observed phenomenon may be regarded as the "force version" of the Coulomb blockade effect.