Extending the photobleaching lifetime in the presence of an optical tweezers by wavefront engineering

Here we lay the foundations for a novel approach to simultaneously performing optical force spectroscopy and single-molecule fluorescence measurements by adapting techniques from wavefront engineering. Our method relies upon the axial displacement (i.e. along the direction of propagation of the trap laser) of an appropriately constructed Laguerre-Gaussian (LG), or 'donut' beam, to generate an optical force on a surface tethered microsphere. With this geometry, one maintains a region of near-infrared (IR) light below the microsphere, where a biomolecule of interest can be placed, that is low enough in intensity to not significantly interfere with fluorescence measurements. We are thus able to separate the fluorescence illumination from the trap light without having to physically separate the two by several micrometers, or employ long DNA linker molecules as handles. As a proof of principal, we show that within the region of low IR intensity, directly below the trap center, the photobleaching lifetime of the organic dye Alexa-647, which is highly sensitive to the intense trap light, is significantly extended.