Detection and Correction of Blinking Bias in Image Correlation Transport Measurements of Quantum Dot Tagged Macromolecules

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic errors caused by fluorescence blinking of the nanoparticles. Temporal correlation analysis of fluorescence microscopy image time series of streptavidin-functionalized (CdSe)ZnS QDs freely diffusing in two dimensions shows that the correlation functions are fit well to a commonly used diffusion decay model, but the transport coefficients can have significant systematic errors in the measurements due to blinking. Image correlation measurements of the diffusing QD samples measured at different laser excitation powers and analysis of computer simulated image time series verified that the effect we observe is caused by fluorescence intermittency. We show that reciprocal space image correlation analysis can be used for mobility measurements in the presence of blinking emission because it separates the contributions of fluctuations due to photophysics from those due to transport. We also demonstrate application of the image correlation methods for measurement of the diffusion coefficient of glycosyl phosphatidylinositol-anchored proteins tagged with QDs as imaged on living fibroblasts

Fast photoluminescence quenching in thin films of 4,4 '-bis(2,2-diphenylvinyl)-1,1 '-biphenyl exposed to air

The photoluminescence (PL) quenching mechanism of UV light and air-exposed amorphous thin films of 4,4'-bis(2,2-diphenylvinyl)-1,1'-biphenyl (DPVBi), a well-known hole-transport material used in organic light-emitting diodes, is studied. Thin films of DPVBi are stable when exposed to UV light in vacuum but tend to degrade if oxygen is present simultaneously. This is evident from the changes in UV-vis absorption spectra of the latter, showing that degradation rate of DPVBi films is linearly proportional to both oxygen concentration and UV light intensity. Mass spectrometry study of such films revealed a number of different oxygen-containing molecules and fragments of DPVBi thus confirming apparent photo-oxidation process. Also, DFT study of molecular DPVBi with and without oxygen was carried out, the IR spectra calculated for the lowest energy molecules found and the results are compared with the experiment. The most sensitive to photo-oxidation is DPVBi photoluminescence, which decays exponentially with respect to the concentration of photo-oxidized DPVBi molecules (impurities). The PL quantum yield of DPVBi thin film drops to a half of its original value for 0.2% of the impurities present, at which point an average distance between DPVBi molecules (the donors) and photo-oxidized DPVBi species (acceptors) is an order of magnitude larger than the separation between two adjacent molecules. This implies a need for a long-range Forster energy transfer, which we rule out based on the lack of a donor-acceptor spectral overlap. The apparent discrepancy can be removed by postulating exciton self-diffusion in DPVBi thin films, for which there is supporting evidence in existing literature

Fast photoluminescence quenching in thin films of 4,4 -bis(2,2-diphenylvinyl)-1,1 -biphenyl exposed to air

The photoluminescence (PL) quenching mechanism of UV light and air-exposed amorphous thin films of 4,4-bis(2,2-diphenylvinyl)-1,1-biphenyl (DPVBi), a well-known hole-transport material used in organic light-emitting diodes, is studied. Thin films of DPVBi are stable when exposed to UV light in vacuum but tend to degrade if oxygen is present simultaneously. This is evident from the changes in UV-vis absorption spectra of the latter, showing that degradation rate of DPVBi films is linearly proportional to both oxygen concentration and UV light intensity. Mass spectrometry study of such films revealed a number of different oxygen-containing molecules and fragments of DPVBi thus confirming apparent photo-oxidation process. Also, DFT study of molecular DPVBi with and without oxygen was carried out, the IR spectra calculated for the lowest energy molecules found and the results are compared with the experiment. The most sensitive to photo-oxidation is DPVBi photoluminescence, which decays exponentially with respect to the concentration of photo-oxidized DPVBi molecules (impurities). The PL quantum yield of DPVBi thin film drops to a half of its original value for 0.2% of the impurities present, at which point an average distance between DPVBi molecules (the donors) and photo-oxidized DPVBi species (acceptors) is an order of magnitude larger than the separation between two adjacent molecules. This implies a need for a long-range Forster energy transfer, which we rule out based on the lack of a donor-acceptor spectral overlap. The apparent discrepancy can be removed by postulating exciton self-diffusion in DPVBi thin films, for which there is supporting evidence in existing literature. (C) 2015 Elsevier B.V. All rights reserved

Investigating hepatitis C virus infection using super-resolution microscopy

Super-resolution microscopy (SRM) can provide a window on the nanoscale events of virus replication. Here we describe a protocol for imaging hepatitis C virus-infected cells using localization SRM. We provide details on sample preparation, immunostaining, data collection, and super-resolution image reconstruction. We have made all efforts to generalize the protocol to make it accessible to all budding super-resolution microscopists