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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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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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Ultrafast processes for bulk modification of transparent materials

K. Itoh, W. Watanabe, S. Nolte, and C. B. Schaffer

Materials Research Society Bulletin (2006)

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When a femtosecond laser pulse is focused inside a transparent material, the optical intensity in the focal volume can become high enough to induce permanent structural modifications such as a refractive index change or the formation of a small vacancy. Thus, one can micromachine structures inside the bulk of a transparent material in three dimensions. We review the mechanisms of and techniques for bulk modification of transparent materials using femtosecond laser pulses and discuss the fabrication of photonic and other structures in transparent materials, including waveguides, couplers, gratings, diffractive lenses, optical data storage, and microfluidic channels.

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Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor

Nan Shen, Dabajyoti Datta, Chris B. Schaffer, Philip LeDuc, Donald E.Ingber, Eric Mazur

Mechanics and Chemistry of Biosystems (2005)

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Analysis of cell regulation requires methods for perturbingmolecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.

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Femtosecond laser-drilled capillary integrated into a microfluidic device

Tyson N. Kim, Kyle Campbell, Alex Groisman, David Kleinfeld, and Chris B. Schaffer

Applied Physics Letters (2005)

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Recent growth in microfluidic technology is, to a large extent, driven by soft lithography, a high-throughput fabrication technique where polymer materials, such as polysdimethyld siloxane sPDMSd, are molded to form microscopic channel networks. Nevertheless, the channel architectures that can be obtained by molding are limited. We address this limitation by using femtosecond laser micromachining to add unmoldable features to the microfluidic devices. We apply laser ablation to drill microcapillaries, with diameters as small as 0.5 mm and aspect ratios as high as 800:1, in the walls of molded PDMS channels. Finally, we use a laser-drilled microcapillary to trap a polystyrene bead by suction and hold it against a shear flow

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Morphology of femtosecond laser-induced structural changes in bulk transparent materials

Chris B. Schaffer, Alan O. Jamison, and Eric Mazur

Applied Physics Letters (2004)

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Using optical and electron microscopy, we analyze the energy and focusing angle dependence of structural changes induced in bulk glass by tightly focused femtosecond laser pulses. We observe a transition from small density variations in the material to void formation with increasing laser energy. At energies close to the threshold for producing a structural change, the shape of the structurally changed region is determined by the focal volume of the objective used to focus the femtosecond pulse, while at higher energies, the structural change takes on a conical shape. From these morphological observations, we infer the role of various mechanisms for structural change

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All-Optical Histology Using Ultrashort Laser Pulses

Philbert S. Tsai, Beth Friedman, Agustin I. Ifarraguerri, Beverly D. Thompson, Varda Lev-Ram, Chris B. Schaffer, Qing Xiong, Roger Y. Tsien, Jeffrey A. Squier, and David Kleinfeld

Neuron (2003)

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As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 uJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100um or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.

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Bulk heating of transparent materials using a high-repetition-rate femtosecond laser

Chris B. Schaffer, Jose F. Garcia, and Eric Mazur

Applied Physics (2003)

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Femtosecond laser pulses can locally induce structural and chemical changes in the bulk of transparent materials, opening the door to the three-dimensional fabrication of optical devices. We review the laser and focusing parameters that have been applied to induce these changes and discuss the different physical mechanisms that play a role in forming them. We then describe a new technique for inducing refractive-index changes in bulk material using a high-repetition-rate femtosecond oscillator. The changes are caused by a localized melting of the material, which results from an accumulation of thermal energy due to nonlinear absorption of the high-repetition-rate train of laser pulses.

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Customization of Poly(dimethylsiloxane) Stamps by Micromachining Using a Femtosecond-Pulsed Laser

Daniel B. Wolfe, Jonathan B. Ashcom, Jeremy C. Hwang, Chris B. Schaffer, Eric Mazur, and George M. Whitesides

Advanced Materials (2003)

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This work describes the use of focused, high-intensity light from a Ti:sapphire laser that generates femtosecond pulses to create topographical structure in a flat surface of poly(dimethylsiloxane) (PDMS), and the use of the PDMS surfaces patterned in surface bas-reliefis the material most widely used for printing and stamping in soft lithography, and a material widely used in microfluidic systems. The bas-relief patterns required in these applications are usually fabricated by casting PDMS against a complementary bas-relief pattern in photoresist, fabricated in turn by photolithography. This process works well, but is not applicable to the preparation of PDMS stamps required for all types of problems; printing on spherical surfaces is an example.

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Microexplosions in tellurite glasses

S.K. Sundaram, Chris B. Schaffer, and Eric Mazur

Applied Physics (2003)

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Femtosecond laser pulses were used to produce localized damage in the bulk and near the surface of baseline, Al2O3-doped and La2O3-doped sodium tellurite glasses. Single or multiple laser pulses were non-linearly absorbed in the focal volume by the glass, leading to permanent changes in the material in the focal volume. These changes were caused by an explosive expansion of the ionized material in the focal volume into the surrounding material, i.e. a microexplosion. The writing of simple structures (periodic array of voxels, as well as lines) was demonstrated. The regions of microexplosion and writing were subsequently characterized using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS) and atomic force microscopy (AFM). Fingerprints of microexplosions (concentric lines within the region and a concentric ring outside the region), due to the shock wave generated during microexplosions, were evident. In the case of the baseline glass, no chemistry change was observed within the region of the microexplosion. However, Al2O3-doped and La2O3-doped glasses showed depletion of the dopant fromthe edge to the center of the region of the microexplosions, indicating a chemistry gradient within the regions. Interrogation of the bulk- and lasertreated regions using micro-Raman spectroscopy revealed no structural change due to the microexplosions and writing within these glasses

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