Precise optical manipulation techniques for exploring neuronal function

Abstract

This thesis presents two optical techniques for manipulating neurons in the analysis of their function, namely time-gated holographic two-photon photostimulation and femtosecond laser dendrotomy. At the start of this thesis, no illumination technique had been able to activate synapses along the three-dimensional (3D) dendritic arbour of a neuron using realistic spatio-temporal patterns of light that resemble physiological stimuli, a prerequisite to a systematic study of synaptic integration. This thesis demonstrates that holographic projection with a spatial light modulator (SLM) enables the generation of multiple uncaging focal spots at arbitrary locations in 3D and allows full control over the spatial organisation of uncaging sites. The incorporation of a digital micromirror device (DMD) in the setup allows high-speed independent switching of individual focal spots, enabling control over the temporal organisation of uncaging events. Together, the SLM-DMD combination offers unprecedented flexibility in the design and generation of spatio-temporal patterns of synaptic input for integration studies. A technique for optimising the holographic projection technique used for photostimulation is also presented. The technique incorporates a generalised phase contrast (GPC) setup into a typical SLM holographic setup to generate a complex field hologram, as opposed to conventional phase-only holograms. Numerical simulations show that the technique produces higher optical throughput for the projection of three or more foci. It also significantly decreases the intensity of unwanted higher diffraction orders, providing cleaner excitation patterns. This technique has the potential to increase the number of holographic photostimulation foci achievable with current techniques. Finally, this thesis demonstrates femtosecond laser dendrotomy as a precise and minimally-invasive way to prune dendrites at different locations in the tree. Its impact on the firing frequency of the neuron is evaluated. This technique has been used \emph{in vivo} but neuronal properties during and after dendrotomy have not been previously measured. This study found that small-scale dendrotomy has negligible effect on the input resistance or the firing pattern of the neuron. However, large-scale dendrotomy significantly increases the input resistance of the neuron and its excitability. This technique offers the capability to experimentally manipulate dendritic morphology for the study of neuronal structure-function relationship. Show more