The DNA double helix challenged by force

The technically challenging field of single-molecule biophysics has established itself in the last decade by granting access to detailed information about the fate of individual biomolecules, unattainable in traditional biochemical assays. The appeal of single-molecules methods lies in the directness of the information obtained from individual biomolecules. Technological improvements in single-molecule methods have made it possible to combine optical tweezers, fluorescence microscopy and microfluidic flow systems. Such a combination of techniques has opened new possibilities to study complex biochemical reactions on the single-molecule level. This chapter provides general considerations for the development of a combined optical trapping, fluorescence microscopy and microfluidics instrument, along with methods to solve technical issues that are critical for designing successful experiments. Finally, several experiments are demonstrated to illustrate the power of this combination of techniques.