This thesis presents three research topics that are connected through the development and application of holographically controlled optical tweezers. (1) Optimization of the diffraction efficiency of a spatial light modulator, which is the diffractive optical element that enables holographic control of the laser beam in three-dimensions. (2) Multiplexed fluorescence spectroscopy of trapped particles. (3) Application of holographic optical tweezers to study the mammalian extra-cellular matrix that is composed primarily of type-I collagen.