Geoffrey M. Lowman

Generation and mixing of subfemtoliter aqueous droplets on demand.

We describe a novel method of generating monodisperse subfemtoliter aqueous droplets on demand by means of piezoelectric injection. Droplets with volumes down to 200 aL are generated by this technique. The droplets are injected into a low refractive index perfluorocarbon so that they can be optically trapped. We demonstrate the use of optical tweezers to manipulate and mix droplets. For example, using optical tweezers we bring two droplets, one containing a calcium sensitive dye and the other calcium chloride, into contact. The droplets coalesce with a resulting reaction time of about 1 ms. The monodispersity, manipulability, repeatability, small size, and fast mixing afforded by this system offer many opportunities for nanochemistry and observation of chemical reactions on a molecule-by-molecule basis.

Nanoscale morphology of polyelectrolyte self-assembled films probed by scanning force and near-field scanning optical microscopy

Abstract We applied shear force microscopy, an analog to attractive-mode atomic force microscopy, and near-field scanning optical microscopy (NSOM) to the study of surface roughness and nanoscale morphology in polyelectrolyte self-assembled layers. Our data show that the surface roughness of multilayer films on glass grows linearly with the number of polyelectrolyte bilayers for the first 10 bilayers, and then asymptotes at a surface roughness of 4 nm. The surface of these films is characterized by bumps of 100–500 nm in diameter and 25–50 nm tall. In addition, the size and density of the bumps for each bilayer are uncorrelated to the previous surface. This result is in sharp contrast to what is observed for other self-assembled layer structures, such as metal-phosphonate and Langmuir–Blodgett self-assembly, where the surface roughness linearly increases with the number of layers. Subsurface morphology in thin films was observed via fluorescence NSOM of a dye-doped polyelectrolyte film. The NSOM images show domains of higher and lower fluorescence intensity, which could be assigned to local changes in the film thickness, suggesting that the dye molecules are uniformly distributed within the film. We also show that both the initial and asymptotic surface roughness can be dramatically decreased if the bare glass surface is treated with a high charge-density polyelectrolyte, such as poly(ethyleneimine) (PEI), prior to the bilayer formation. Finally, we observed that if the initial substrate is rough, growth of the polyelectrolyte layers acts to smooth the surface until the equilibrium value is reached.

Probing nanoscale photo-oxidation in organic films using spatial hole burning near-field scanning optical microscopy

Spatial hole burning near-field scanning optical microscopy (SHB–NSOM) is used to locally photopattern three species of organic thin films, poly(2-methoxy, 5-(2′-ethyl hexyloxy)–p-phenylene vinylene) (MEH–PPV), tris-8-hydroxyquinoline aluminum (Alq3) and dye-functionalized polyelectrolyte self-assembled layers, on a 100 nm length scale. In SHB–NSOM the film is illuminated with light from a stationary NSOM tip to induce photo-oxidation. The reduction in the fluorescence yield resulting from this exposure is then mapped using fluorescence NSOM (FL–NSOM). We have examined the localized photo-oxidation as a function of time, position, and environment free from the limits of far-field spatial averaging. In all of the thin film materials studied we find that the long-time diameter of the dark spot is much larger than the tip diameter and is a signature of energy migration. Characteristic lengths of the energy migration are extracted from this data by a simple diffusion model and are found to be of the order of ...

Green fluorescent protein in inertially injected aqueous nanodroplets.

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 +/- 0.1 ns at 3 microM and 14.0 +/- 0.2 ns at 10 microM. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 +/- 1.0 ns at 3 microM and 15.5 +/- 1.6 ns at 10 microM. We conclude that the rotational motion inside the nanodroplets is consistent with rotation in free solution and that the protein therefore does not aggregate at the water-oil interface. Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.

Hydrosomes: femtoliter containers for fluorescence spectroscopy studies

We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary.