Tetramethylrhodamine, single-molecule

Deniz etal. [82 ] applied spFRET in one of the first studies that demonstrated its use as a potential structural probe revealing heterogeneity of proteins in solution. Chymotrypsin inhibitor 2 (CI2) was FRET labelled with the dye pair TMR (tetramethylrhodamine) and Cy5 (see Chapter 4). Single molecule diffusion experiments were then performed as a function of chemical denaturant. The folded and unfolded subpopulations of the protein at various denaturant concentrations were resolved and the populations of each state (estimated by the relative area of each peak) were shown to be in broad agreement with the supporting ensemble measurements. Further, changes were seen in the mean FRET efficiency of the unfolded distribution with increasing denaturant. 

Fig. Ila-c Spectral fluctuations of single tetramethylrhodamine (TMR)-labeled myosin molecules a fluorescence images of single TMR-myosin molecules. Arrow represents the fluorescence spot analyzed b a series of fluorescence spectra from a single TMR-myosin arrow in a) with time. Spectra were taken sequentially every 0.61 s with the collection time of 0.5 s for each spectrum c fluctuations in the fluorescence spectrum of single TMR-myosin. The spectral mean was plotted as a function of time, inset) Total fluorescence intensity obtained by integrating fluorescence intensity for wavelengths from 540 to 600 nm for each spectrum...

Fig. 15 An example of the blinking measured from a single tetramethylrhodamine molecules. The fluorescence intensity was measured with an avalanche photodiode single photon counting module. Excitation wavelength, 514.5 nm. Sampling interval, 10 ms...

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