Fig. 1
Experimental workflow for single-molecule imaging and tracking. (a) Genetic engineering of yeast strains for visualizing the single-molecules of chromatin-bound histone H3 (Hht1) and free GFP. The halotag was fused at the C-terminus of the hht1 gene endogenously by genetic engineering using the hygromycin resistance (HygR) gene as a selection marker. The recombinant gene hht1-halotag is expressed from its native promoter. Similarly, the halotag was fused at the N-terminus of the GFP gene in a cloned vector (with the URA4 gene as a selection marker), and expressed from the nmt1 promoter. nls = nuclear localization signal sequence. (b) Cells are taken out from the glycerol stocks (from −80° C freezer) and grown to the log phase before imaging. The Hht1-HaloTag protein was sparsely labeled with a JF646-HTL so that only 1 to 5 molecules per nucleus were labeled. (c) Single-molecule imaging is performed using fast and slow imaging regimes (time-lapse movies with 15 ms time intervals for 1000 frames and 200 ms time intervals for 200 frames, respectively). (d) Using tracking software (MatlabTrack and TrackIt), the single molecules are detected based on intensity thresholding, and the trajectories are made by connecting particles from one frame to the next frame. (e) Based on the X- and Y- positions of the molecules in their trajectories and how long the molecules survive, the biophysical parameters (such as jump distances, mean square displacement, diffusion coefficient, fraction of bound and free molecules, and residence time) can be quantified.
