The advent of ultrashort (femtosecond) laser light pulses as laboratory tool opens up new opportunities to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon microscopy. More recently, high-fluence ultrashort pulses have been exploited to reliably create micron-sized ablations in brain tissue with minimal collateral damage. These ablations are the driving technology in an all-optical histology, which allows anatomy to be imaged with micrometer resolution throughout the brain. These ablations can also be used to perturb neocortical blood flow as a means to probe normal and diseased tissues. Yet much additional effort is required to use and advance the mixture of multi- photon ablation and imaging techniques as a means to enable studies of neuronal and vascular architectonics. Our proposed instrumentation concerns the use of ultrashort laser pulses for focal ablation at high fluence (energy per area), and imaging, at lower fluence, to address open issues in neuroanatomy and neurovascular coupling. Two synergistic applications will serve as test beds for our technical goals: ? Establish all-optical based histology as a standard anatomical tool. This includes the optimization of parameters and the correction of spherical aberration for both ablation and imaging. We proposed to reconstruct neuronal and non-neuronal soma and vasculature positions throughout rat vibrissa sensory cortex and to reconstruct mitochondrial density and vasculature throughout vibrissa sensory cortex. ? Advance the optical induction and monitoring of targeted vascular blocks. We will perturb blood flow in connective arteriol networks as well as deep capillary networks to study flow dynamics in different angioarchitectures. The proposed advancement in the breadth of nonlinear optical methods will provide a novel tool for manipulating and probing tissues that is simultaneously imaged with two-photon microscopy. We will make these tools reliable and readily available to the biomedical community. The proposed model systems may substantially improve upon our understanding of brain architectonics and stroke formation. That may lead to improvements in preclinical models to assay therapeutics for stroke. 3ERFORMANCESITE(S) (organization, city, state) Department of Physics University of California at San Diego La Jolla, CA 92093-0319 KEY PERSONNELSeeinstructionson Page 11. Usecontinuationpagesas neededto providethe required information inthe format shown below. Name Organization Role on Project Kleinfeld, David Physics Dept, UCSD Principal Investigator Dolnick, Earl M. Physics Dept., UCSD Engineer Klelnfeld, David Type the name of the principal investigator/program director at the top of each printed page and each continuation page. (Fortype specifications, see instructions on page6.) ; RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,
| Shih, Andy Y; Rühlmann, Charlotta; Blinder, Pablo et al. (2015) Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture. Microcirculation 22:204-218 |
| Driscoll, Jonathan D; Shih, Andy Y; Drew, Patrick J et al. (2013) Two-photon imaging of blood flow in the rat cortex. Cold Spring Harb Protoc 2013:759-67 |
| Jeong, Diana C; Tsai, Philbert S; Kleinfeld, David (2012) Prospect for feedback guided surgery with ultra-short pulsed laser light. Curr Opin Neurobiol 22:24-33 |
| Driscoll, Jonathan D; Shih, Andy Y; Iyengar, Satish et al. (2011) Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy. J Neurophysiol 105:3106-13 |
| Kleinfeld, David; Bharioke, Arjun; Blinder, Pablo et al. (2011) Large-scale automated histology in the pursuit of connectomes. J Neurosci 31:16125-38 |
| Drew, Patrick J; Shih, Andy Y; Driscoll, Jonathan D et al. (2010) Chronic optical access through a polished and reinforced thinned skull. Nat Methods 7:981-4 |
| Drew, Patrick J; Blinder, Pablo; Cauwenberghs, Gert et al. (2010) Rapid determination of particle velocity from space-time images using the Radon transform. J Comput Neurosci 29:5-11 |
| Tsai, Philbert S; Blinder, Pablo; Migliori, Benjamin J et al. (2009) Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems. Curr Opin Biotechnol 20:90-9 |
| Devor, Anna; Hillman, Elizabeth M C; Tian, Peifang et al. (2008) Stimulus-induced changes in blood flow and 2-deoxyglucose uptake dissociate in ipsilateral somatosensory cortex. J Neurosci 28:14347-57 |
| Devor, Anna; Tian, Peifang; Nishimura, Nozomi et al. (2007) Suppressed neuronal activity and concurrent arteriolar vasoconstriction may explain negative blood oxygenation level-dependent signal. J Neurosci 27:4452-9 |
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