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Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

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This article describes the design and development of squaraine-based semiconducting polymer dots (Pdots) that show large Stokes shifts and narrow-band emissions in the near-infrared (NIR) region. Fluorescent copolymers containing fluorene and squaraine units were synthesized and used as precursors for preparing the Pdots, where exciton diffusion and likely through-bond energy transfer led to highly bright and narrow-band NIR emissions. The resulting Pdots exhibit the emission full width at half-maximum of ∼36 nm, which is ∼2 times narrower than those of inorganic quantum dots in the same wavelength region (∼66 nm for Qdot705). The squaraine-based Pdots show a high fluorescence quantum yield (QY) of 0.30 and a large Stokes shift of ∼340 nm. Single-particle analysis indicates that the average per-particle brightness of the Pdots is ∼6 times higher than that of Qdot705. We demonstrate bioconjugation of the squaraine Pdots and employ the Pdot bioconjugates in flow cytometry and cellular imaging applications. Our results suggest that the narrow bandwidth, high QY, and large Stokes shift are promising for multiplexed biological detections.

In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.