The advent of ultrashort laser light pulses as a laboratory tool has provided the opportunity to probe and manipulate structure and function in biological systems. This award supports development of an instrument that will use nonlinear absorption to create micrometer-sized ablations in nervous tissue, with minimal collateral damage. The instrument can be integrated with existing nonlinear imaging devices, principally the multi-photon microscope. Prior work has shown that ultrashort laser pulses can be used for an all-optical approach to histology in which anatomical structure with micrometer resolution is obtained without the use of mechanical sectioning devices. Pilot data has also demonstrated that such pulses can block flow in specific, targeted blood vessels, thus enabling studies of the biofluid mechanics of the vessels. The proposed instrument development program will design and realize ultrashort pulsed laser technology capable of a mixture of optical ablation and nonlinear optical imaging. The effort will result in a near turn-key system that uses commercially available laser oscillators and regenerative amplifiers, together with a mixture of commercial and custom opto-mechanical and electronic components and software. The expected applications include the automatic anatomical mapping of neuronal and non-neuronal nuclei, vasculature, and subcellular structures within the rodent brain, and the optical induction of axonomy in which axons in the leech ganglion are cut to study circuit dynamics in the swim command network. Additional potential applications include submicrometer surgery to ablate organelles, and transient disruption of the blood-brain barrier. The design and realization of the instrument will expose students to use of state-of the art optical instrumentation for the pursuit of biological questions. The proposed work will specifically train two postdoctoral researchers in instrumentation and experimentation at the optics/biology interface.
