A new technique for reversible permeabilization of live cells for intracellular delivery of quantum dots.

A major challenge with the use of quantum dots (QDs) for cellular imaging and biomolecular delivery is the attainment of QDs freely dispersed inside the cells. Conventional methods such as endocytosis, lipids based delivery and electroporation are associated with delivery of QDs in vesicles and/or as aggregates that are not monodispersed. In this study, we demonstrate a new technique for reversible permeabilization of cells to enable the introduction of freely dispersed QDs within the cytoplasm. Our approach combines osmosis driven fluid transport into cells achieved by creating a hypotonic environment and reversible permeabilization using low concentrations of cell permeabilization agents like Saponin. Our results confirm that highly efficient endocytosis-free intracellular delivery of QDs can be accomplished using this method. The best results were obtained when the cells were treated with 50 µg ml⁻¹ Saponin in a hypotonic buffer at a 3:2 physiological buffer:DI water ratio for 5 min at 4 °C.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage.

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.

Localized delivery of DNA to the cells by viral collagen-loaded silica colloidal crystals.

Low-molecular-weight colloidal crystals with enhanced biocompatibility and ordered porous structure are used in drug-delivery systems. The objective of our study is to demonstrate the use of silica nanoscale colloid particles for localized recombinant DNA release. The colloids were coated with collagen-containing viral vector constructs of lentiviral green fluorescent protein (GFP), and solidified at 37 degrees C. The colloid-collagen-viral vector platform (CCP) was transferred to cell monolayer cultures of human lung fibroblasts. Results show specific infection of cells directly beneath the platform, as evidenced by positive GFP in their cytoplasm, while neighboring cells show no cytoplasmic GFP The infection of specific cells is probably due to the gradual release of viral particles from the collagen matrix by cell-secreted collagenase, which avoids overdosing the cells with viral particles, resulting from the cytopathic effect often seen with high-titer viral infection. Cells infected with the lentiviral-GFP or lentivirus alone, not incorporated into the colloid-collagen device, show caspase 3-associated apoptotic cell death. This suggests that colloidal crystal-coated collagen may be used as a powerful platform to deliver genes of choice to localized subgroups of specific cells of interest. This specificity in the delivery mode is beneficial for functional studies of gene-directed impact on a particular cell population of interest in a heterogeneous cell culture.

Observation of the triplet exciton in EuS-coated single-walled nanotubes.

Photon absorption by carbon nanotubes creates bound electron-hole pairs called excitons, which can exist in spin-polarized triplet or spin-unpolarized singlet configurations. Triplet excitons are optically inactive owing to the weak spin-orbit coupling in nanotubes. This prevents the optical injection of electron spin into nanotubes for spintronic applications and limits the efficiency of photocurrent generation. Here, we show that it is possible to optically excite the triplet exciton by using a ferromagnetic semiconductor as a spin filter to mix the singlet and triplet excitons. The triplet contribution to the photocurrent is detected, representing the first direct evidence of the triplet exciton in carbon nanotubes.

Exciton dissociation and stark effect in the carbon nanotube photocurrent spectrum.

The field-dependent photocurrent spectrum of individual carbon nanotubes is measured using a displacement photocurrent technique. A series of peaks is observed in the photocurrent corresponding to both excitonic and free carrier transitions. The photocurrent peak corresponding to the ground state exciton increases by a factor of 200 beyond a critical electric field, and shows both red and blue shifts depending on the field regime. This provides evidence for field-induced mixing between excitonic and free carrier states.

Strain tuning of the photocurrent spectrum in single-wall carbon nanotubes.

The effect of uniaxial strain on the photocurrent spectrum of semiconducting single-wall carbon nanotubes is measured. The energy of the lowest-lying free electron transition is observed to shift with strain as predicted by a simple noninteracting model. The higher-order transitions also shift with strain, but being excitonic, their strain dependence differs from the predictions for the free carrier states. An anomalous photocurrent increase is also observed near the ground-state transition and is attributed to the formation of optically active defect states within the nanotube band gap.

Field-enhanced photocurrent spectroscopy of excitonic states in single-wall carbon nanotubes.

Excitonic and free-carrier transitions in single-wall carbon nanotubes are distinguished using field-enhanced photocurrent spectroscopy. Electric field dissociation allows for the detection of bound-exciton states that otherwise would not contribute to the photocurrent. Excitonic states associated with both the ground-state semiconductor and the ground-state metallic nanotube transitions are resolved. The observation of a metallic excitonic state corroborates recent predictions of a symmetry gap existing in metallic nanotubes.

Mechanisms of 1D crystal growth in reactive vapor transport: indium nitride nanowires.

Indium nitride (InN) nanowire synthesis using indium (In) vapor transport in a dissociated ammonia environment (reactive vapor transport) is studied in detail to understand the nucleation and growth mechanisms involved with the so-called "self-catalysis" schemes. The results show that the nucleation of InN crystal occurs first on the substrate. Later, In droplets are formed on top of the InN crystals because of selective wetting of In onto InN crystals. Further growth via liquid-phase epitaxy through In droplets leads the growth in one dimension (1D), resulting in the formation of InN nanowires. The details about the nucleation and growth aspects within these self-catalysis schemes are rationalized further by demonstrating the growth of heteroepitaxially oriented nanowire arrays on single-crystal substrates and "tree-like" morphologies on a variety of substrates. However, the direct nitridation of In droplets using dissociated ammonia results in the spontaneous nucleation and basal growth of nanowires directly from the In melt surface, which is quite different from the above-mentioned nucleation mechanism with the reactive vapor transport case. The InN nanowires exhibit a band gap of 0.8 eV, whereas the mixed phase of InN and In(2)O(3) nanowires exhibit a peak at approximately 1.9 eV in addition to that at 0.8 eV.