We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 milliseconds to follow three dimensional molecular motion. This method has substantial advantages over 3D molecular tracking methods based upon CCD cameras, including increased Z tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon anti-bunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatio-temporal dynamics of live cells, we follow individual QD-labeled IgE receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three dimensional, nano-scale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell. KeywordsQuantum Dot; Single Molecule; Fluorescence; Tracking; Microscopy; Cell The direct observation of individual biological molecules in motion can transform our view of important biophysical and cellular processes. 1 For example, single molecule tracking has shed significant light on cellular membrane dynamics 2-4 , motor protein kinetics 5,6 , and gene regulation 7 . Advantages of a single molecule approach include the ability to observe dynamic, stochastic behavior (such as compartmentalized diffusion 2, 4 ) that would be masked in ensemble measurements and the ability for localization of molecules with a precision well below the diffraction limit of light 5,6 . To date the field has primarily relied * Corresponding Author: jwerner@lanl.gov. 8,16 or follow the Z position with multiple cameras or image planes 9, 14 . While these camera-based techniques can capture 3D molecular motion, they are generally limited in their Z-tracking range in cells to approximately plus or minus one μm from a fixed focal plane 8,10,14,17 , limited by the shallow depth of field of high numerical aperture microscope objectives needed for single molecule work. We point out the obvious: many cells are substantially thicker than two microns and different methods and techniques are required to follow single molecules throughout entire three dimensional cell volumes. In addition to its quite limiting Z-tracking range, CCD-based tracking approaches are also bounded in temporal resolution by the CCD frame rate (~1 ms for fast EM-CCDs), and must illuminate an entire cell slice at relatively large excitation intensities (~40 W/cm 2 ).In contrast to ...

Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.

While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or ‘blinking,’ which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking ‘giant’ quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.