Abstract
Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.
Highlights
The last two decades have seen a dramatic increase in the utilization of single molecule detection methods in a variety of biological applications [1,2,3,4]
The results for IQDon and FQDon for each quantum dots (QDs) color and experimental condition are summarized in Fig. 3 and in detail in Table S2.The results show that there is quantitative difference in IQDon among the different colors of QDs where the order, ranked in order of decreasing IQDon, was IQD625on
We have not accounted for the spectral dependence of the quantum efficiency (QE) of the CCD camera (Fig. S2) which in this case would result in an increase of the absolute brightness of the QD705s by
Summary
The last two decades have seen a dramatic increase in the utilization of single molecule detection methods in a variety of biological applications [1,2,3,4]. Spatio-temporal investigations using SPT are particular prone to artifacts due to e.g. the probe size and valency [6,7,8] This is because SPT has historically been performed with antibody conjugated colloidal gold particles [9,10] that are large (RH
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