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Combination of double optical tweezers with highly sensitive fluorescence

par Maxime Dahan - 23 octobre 2007

For some fluorescence investigations it is feasible to allow for a controlled positioning of targets within the field of view. To this end, we built a modfied fluorescence set-up that additionally incorporates double optical tweezers. Briefly, in an optical tweezer, a transparent particle in the µm-range can be held by the radiation pressure of a tightly focussed beam of a high intensity infrared laser. By splitting the light into two beams which can be moved independently by a set of lenses and mirrors, one obtains a tool for simultaneous micromanipulation of two beads. The beads can be functionalized with streptavidin to bind the DNA. Several adaptions to the optics are necessary to combine the optical tweezers with fluorescence microscopy. Hence, the intense IR light has to be prevented from interfering with the fluorescence detection using a high efficient blocking filter in the IR. It has to be also considered that the fluorescence excitation has to be accomplished by epifluorescence microscopy which facilitates excitation remote from the surface, but produces a higher background signal compared to TIRFM. Although this is in part compensated by the effect that the main background source stems from the surface, this requires the application of semiconductor nanocrystals as fluorescence source. Although this design is optimized to be used for stretching DNA, it can be used to control positioning of any molecules or particles which either can be immobilized to transparent beads or display a size in the µm range and are transparent themselves suchas cells or bacteria.

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