Optical tweezers and laser scissors are invaluable tools to manipulate cells on the micro and nano scale level. By integrating laser technologies with imaging and biochemical techniques, robust systems can be created to study various biological and biochemical processes. The purpose of this thesis is to describe two different optical methods used to study two cell-based problems. In the first experiment, optical tweezers are used to examine the effects of viscosity on sperm motility and energetics using a 1064nm Nd:YVO₄ continuous laser. In the second experiment, laser scissors are used to study growth cone response from laser induced damage using a Vanguard Nd: YVO₄ second harmonic generator (SHG) 532 nm picosecond green laser. The proceeding sections describe the optical systems for each experiment as well as the methods used. Results of both studies are presented and discussed in detail

By focusing a laser with short pulses to a diffraction-limited spot, single nerve axons can be precisely targeted and injured. Subsequent repair can be analyzed using various imaging and biochemical techniques to understand the repair process. In this chapter, we will describe a robotic laser microscope system used to injure nerve axons while simultaneously observing repair using phase and fluorescence microscopy. We provide procedures for controlled laser targeting and an experimental approach for studying axonal repair in embryonic rat hippocampus neurons.

Axonal injury and stress have long been thought to play a pathogenic role in a variety of neurodegenerative diseases. However, a model for studying single-cell axonal injury in mammalian cells and the processes of repair has not been established. The purpose of this study was to examine the response of neuronal growth cones to laser-induced axonal damage in cultures of embryonic rat hippocampal neurons and induced pluripotent stem cell (iPSC) derived human neurons. A 532-nm pulsed [Formula: see text] picosecond laser was focused to a diffraction limited spot at a precise location on an axon using a laser energy/power that did not rupture the cell membrane (subaxotomy). Subsequent time series images were taken to follow axonal recovery and growth cone dynamics. After laser subaxotomy, axons thinned at the damage site and initiated a dynamic cytoskeletal remodeling process to restore axonal thickness. The growth cone was observed to play a role in the repair process in both hippocampal and iPSC-derived neurons. Immunofluorescence staining confirmed structural tubulin damage and revealed initial phases of actin-based cytoskeletal remodeling at the damage site. The results of this study indicate that there is a repeatable and cross-species repair response of axons and growth cones after laser-induced damage.