biomolecules with up to zeptomole sensitivity in bulk experiments 7,8,13 . Moreover, the binding of proteins to single functionalized particles can be followed by dark-field microscopy, by exploiting the dependence of the plasmon resonance wavelength on the refractive index of the particle's surroundings 14,15 . The plasmon resonance wavelength of a metal nanoparticle is also affected by other nanoparticles that are in its immediate environment. When two nanoparticles are brought into proximity, their plasmons couple, which shifts the resonance wavelength depending on the particle separation. Since this effect is well known theoretically 16, 17 and experimentally observed for fixed distances 18, 19 , we sought to explore its use as molecular ruler.We applied this 'plasmon ruler' to study the dynamics of DNA hybridization on a single We employed 40 nm diameter gold and silver nanoparticles. The particle diameter was chosen to ensure a sufficiently intense light scattering signal while minimizing any effects of the particles on proximal biomolecules. However, we subsequently found that a reduction in particle size to 20 nm for silver and 30 nm for gold is possible with the current technique.Particles were illuminated with unpolarized white light and light scattered by individual particles was collected by a darkfield microscope in transmission mode (Fig. 1a) 25 . Upon introduction of streptavidin-functionalized particles into the BSA-biotin coated glass chamber, we immediately observed numerous scattering sources adhering to the chamber surfaces. Nanoparticles were vividly colored: individual silver nanoparticles were blue (Fig. 1b), gold nanoparticles were green ( Fig. 1c), aggregates were red-shifted compared to individual particles (typically by about 50 nm for gold, 150nm for silver), and dust and scratches were white.Our first application of plasmon coupling was to monitor the directed assembly of functionalized particle pairs. We used the surface immobilized particles (Figs. 1b and c) as anchors for single stranded DNA (ssDNA) functionalized particles. The 33-nucleotide ssDNA molecules had a biotin at their 3' end, allowing them to bind to the streptavidin coated anchor particles (Fig. 1a). Shortly after introducing the ssDNA-functionalized particles into the chamber, some scattering centers suddenly changed color due to dimer formation. Silver particles turned from blue to green (Fig. 1b), gold particles turned from green to orange (Fig. 1c). The spectral shift upon dimer formation is considerably larger for the silver particles (102 nm) than for gold particles (23 nm, Fig. 1d). The fraction of surface immobilized particles that captured a ssDNAfunctionalized particle ranged from 10% to 86 % depending on the time the samples had been stored, with fresher particles performing better. Aggregates of more than two particles were easily identified by their intensity and distinct color with multiple peaks in the spectrum (see for example the purple dot in Fig 1b). To avoid these aggregates of more than two partic...

Recent advances in statistical mechanical theory can be used to solve a fundamental problem in experimental thermodynamics. In 1997, Jarzynski proved an equality relating the irreversible work to the equilibrium free energy difference, DeltaG. This remarkable theoretical result states that it is possible to obtain equilibrium thermodynamic parameters from processes carried out arbitrarily far from equilibrium. We test Jarzynski's equality by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations. Application of this equality to the irreversible work trajectories recovers the DeltaG profile of the stretching process to within k(B)T/2 (half the thermal energy) of its best independent estimate, the mean work of reversible stretching. The implementation and test of Jarzynski's equality provides the first example of its use as a bridge between the statistical mechanics of equilibrium and nonequilibrium systems. This work also extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments.

Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg2+) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive Luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated, macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.