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Deep tissue multiphoton microscopy using longer wavelength excitation

Demirhan Kobat, Michael E. Durst, Nozomi Nishimura, Angela W. Wong, Chris B. Schaffer, and Chris Xu

Optics Express (2009)

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We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 µm are performed.

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Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery

Conor P. Foley, Nozomi Nishimura, Keith B. Neeves, Chris B. Schaffer, and William L. Olbricht

Biomed Microdevices (2009)

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Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.

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Penetrating arterioles are a bottleneck in the perfusion of neocortex

Nozomi Nishimura, Chris B. Schaffer, Beth Friedman, Patrick D. Lyden, and David Kleinfeld

Proceedings of the National Academy of Sciences (2007)

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Penetrating arterioles bridge the mesh of communicating arterioles on the surface of cortex with the subsurface microvascular bed that feeds the underlying neural tissue. We tested the conjecture that penetrating arterioles, which are positioned to regulate the delivery of blood, are loci of severe ischemia in the event of occlusion. Focal photothrombosis was used to occlude single penetrating arterioles in rat parietal cortex, and the resultant changes in flow of red blood cells were measured with two-photon laser-scanning microscopy in individual subsurface microvessels that surround the occlusion. We observed that the average flow of red blood cells nearly stalls adjacent to the occlusion and remains within 30% of its baseline value in vessels as far as 10 branch points downstream from the occlusion. Preservation of average flow emerges 350 µm away; this length scale is consistent with the spatial distribution of penetrating arterioles. We conclude that penetrating arterioles are a bottleneck in the supply of blood to neocortex, at least to superficial layers.

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Laser photoablation: a new biomedical tool

Nozomi Nishimura, Chris Schaffer, Beth Friedman, Philbert Tsai, Patrick Lyden, and David Kleinfeld

SPIE Newsroom (2006)

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Ultrashort laser pulses can be used to produce lesions in single blood vessels located in the cortex of live rats, thus enabling the study of microstrokes.

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Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke

Nozomi Nishimura, Chris B. Schaffer, Beth Friedman, Philbert S. Tsai, Patrick D. Lyden, and David Kleinfeld

Nature Methods (2006)

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We present a method to produce vascular disruptions within rat brain parenchyma that targets single microvessels. We used two-photon microscopy to image vascular architecture, to select a vessel for injury and to measure blood-flow dynamics. We irradiated the vessel with high-fluence, ultrashort laser pulses and achieved three forms of vascular insult. (i) Vessel rupture was induced at the highest optical energies; this provides a model for hemorrhage. (ii) Extravasation of blood components was induced near the lowest energies and was accompanied by maintained flow in the target vessel. (iii) An intravascular clot evolved when an extravasated vessel was further irradiated. Such clots dramatically impaired blood flow in downstream vessels, in which speeds dropped to as low as ~10% of baseline values. This demonstrates that a single blockage to a microvessel can lead to local cortical ischemia. Lastly, we show that hemodilution leads to a restoration of flow in secondary downstream vessels.

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 Supplement 3

Two-photon imaging of cortical surface microvessels reveals a robust redistribution of blood flow after vascular occlusion

Chris B. Schaffer, Beth Friedman, Nozomi Nishimura, Lee F. Schroeder, Philbert S. Tsai, Ford F. Ebner, Patrick D. Lyden, and David Kleinfeld

Public Library of Science Biology (2006)

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A highly interconnected network of arterioles overlies mammalian cortex to route blood to the cortical mantle. Here we test if this angioarchitecture can ensure that the supply of blood is redistributed after vascular occlusion. We use rodent parietal cortex as a model system and image the flow of red blood cells in individual microvessels. Changes in flow are quantified in response to photothrombotic occlusions to individual pial arterioles as well as to physical occlusions of the middle cerebral artery (MCA), the primary source of blood to this network. We observe that perfusion is rapidly reestablished at the first branch downstream from a photothrombotic occlusion through a reversal in flow in one vessel. More distal downstream arterioles also show reversals in flow. Further, occlusion of the MCA leads to reversals in flow through approximately half of the downstream but distant arterioles. Thus the cortical arteriolar network supports collateral flow that may mitigate the effects of vessel obstruction, as may occur secondary to neurovascular pathology.

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Minimally disruptive laser-induced breakdown in water

Eli N. Glezer, Chris B. Schaffer, Nozomi Nishimura, and Eric Mazur

Optics Letters (1997)

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We produce minimally disruptive breakdown in water by using tightly focused 100-fs laser pulses and demonstrate the potential use of this technique in microsurgery of the eye. Using time-resolved imaging and piezoelectric pressure detection, we measure the magnitude and speed of propagation of the pressure wave produced in the breakdown. Compared with breakdown with longer pulses, here there is a much lower energy threshold for breakdown of 0.2 mJ , a smaller shock zone diameter (11 mm for 1-mJ pulses), and consistent energy deposition.

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