Interlaced Optical Force-Fluorescence Spectroscopy (IOFF)
Combined Optical Trapping and Single Molecule FRET
Optical tweezers force microscopy provides the ability to manipulate single molecules, offering the ability to measure nanometer scale mechanical interactions or direct access to the molecular reaction coordinate. On the other hand, single molecule fluorescence yields structural details that are either separate or complementary from tweezer measurements.There is great opportunity in using these tools in reporting on complex formation and in general how molecular and cellular machinery responds under loads and also in the development of fluorescence based force sensors.
Combining these two techniques has provided a significant challenge over the past decade, as the high intensity optical trapping beam accelerates photobleaching rates to unacceptably short timescales. This effect, which has been explored in the literature, is especially pronounced with common single moleculefluorophores like Cy3 and Alexa 555.
We developed an approach to minimize this effect that extends fluorescnece emission times to over a minute - plenty of time to do a force experiment! This technique, which we call IOFF, is described here.
The paper describes the details of this experiment, but you can scroll down for another example showing an IOFF measurement of DNA unzipping and our demonstration of combined trapping and single molecule FRET described here.
