Rat embryonic hippocampus and induced pluripotent stem cell derived cultured neurons recover from laser-induced subaxotomy.

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.

Laser microbeam targeting of single nerve axons in cell culture.

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.

Effects of viscosity on sperm motility studied with optical tweezers.

The purpose of this study is to analyze human sperm motility and energetics in media with different viscosities. Multiple experiments were performed to collect motility parameters using customized computer tracking software that measures the curvilinear velocity (VCL) and the minimum laser power (Pesc) necessary to hold an individual sperm in an optical trap. The Pesc was measured by using a 1064 nm Nd:YVO(4) continuous wave laser that optically traps motile sperm at a power of 450 mW in the focused trap spot. The VCL was measured frame by frame before trapping. In order to study sperm energetics under different viscous conditions sperm were labeled with the fluorescent dye DiOC(6)(3) to measure membrane potentials of mitochondria in the sperm midpiece. Fluorescence intensity was measured before and during trapping. The results demonstrate a decrease in VCL but an increase in Pesc with increasing viscosity. Fluorescent intensity is the same regardless of the viscosity level indicating no change in sperm energetics. The results suggest that, under the conditions tested, viscosity physically affects the mechanical properties of sperm motility rather than the chemical pathways associated with energetics.