Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via...

We report a method to increase the resolution of single pair fluorescence resonance energy transfer (spFRET) measurements in aqueous solutions. Solution-based spFRET measurements of fluorescently labeled biological molecules (proteins, RNA, DNA) are often used to obtain histograms of molecular conformation without resorting to sample immobilization. However, for solution-phase spFRET studies, the number of photons detected from a single molecule as it diffuses through an open confocal volume element are quite limited. An "average" transit may yield on the order of 40 photons. Shot noise on the number of detected photons substantially limits the resolution of the measurement. The method reported here uses a hydrodynamically focused sample stream to ensure molecules traverse the full width of an excitation laser beam. This substantially increases the average number of photons detected per molecular transit (approximately 85 photons/molecule), which increases measurement precision. In addition, this method minimizes another source of heterogeneity present in diffusive measures of spFRET: the distribution of paths taken through the excitation laser beam. We demonstrate here using a FRET labeled protein sample (a FynSH3 domain) that superior resolution (a factor of approximately 2) can be obtained via molecular cytometry compared to spFRET measurements based upon diffusion through an open confocal volume element.