Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulus-induced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal.

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.

Competing nonlinear optical effects are involved in the interaction of femtosecond laser pulses with transparent dielectrics: supercontinuum generation and multiphoton-induced bulk damage. We measured the threshold energy for supercontinuum generation and bulk damage in fused silica using numerical apertures (NAs) ranging from 0.01 to 0.65. The threshold for supercontinuum generation exhibits a minimum near 0.05 NA and increases quickly above 0.1 NA. For NAs greater than 0.25, we observe no supercontinuum generation. The extent of the blue broadening of the supercontinuum spectrum decreases significantly as the NA is increased from 0.01 to 0.08, showing that weak focusing is important for generating the broadest supercontinuum spectrum. Using a light-scattering technique to detect the onset of bulk damage, we confirmed bulk damage at all NAs studied. At a high NA, the damage threshold is well below the critical power for self-focusing.

A method for noninvasively determining blood oxygenation in individual vessels inside bulk tissue would provide a powerful tool for biomedical research. We explore the potential of coherent anti-Stokes Raman scattering (CARS) spectroscopy to provide this capability. Using the multiplex CARS approach, we measure the vibrational spectrum in hemoglobin solutions as a function of the oxygenation state and observe a clear dependence of the spectral shape on oxygenation. The direct extraction of the Raman line shape from the CARS data using a maximum entropy method phase retrieval algorithm enables quantitative analysis. The CARS spectra associated with intermediate oxygenation saturation levels can be accurately described by a weighted sum of the fully oxygenated and fully deoxygenated spectra. We find that the degree of oxygenation determined from the CARS data agrees well with that determined by optical absorption. As a nonlinear optical technique, CARS inherently provides the 3-D imaging capability and tolerance to scattering necessary for biomedical applications. We discuss the challenges in extending the proof of principle demonstrated to in vivo applications.

We report on third-harmonic generation (THG) of biomolecular solutions at the fluid/glass interface as a means to probe resonant contributions to their nonlinear absorption spectra that could serve as contrast mechanisms for functional imaging. Our source was 100 fs laser pulses whose center wavelength varied from 760 to 1000 nm. We find evidence of a two-photon resonance in the ratio of third-order susceptibilities, Xsample(3w)/Xglass, for aqueous solutions of Rhodamine B, Fura-2, and hemoglobin and a three-photon resonance in Xsample(3w)/Xglass for solutions of bovine serum albumin. Consistent with past work, we find evidence of a one-photon resonance of Xsample(3w)/Xglass for water, while confirming a lack of resonant enhancement for benzene. At physiological concentrations, hemoglobin in different ligand-binding states could be distinguished on the basis of features of its THG spectrum.

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.

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.

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.

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.

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