Chris B. Schaffer

Micromachining bulk glass by use of femtosecond laser pulses with nanojoule energy

Using tightly focused femtosecond laser pulses of just 5 nJ, we produce optical breakdown and structural change in bulk transparent materials and demonstrate micromachining of transparent materials by use of unamplified lasers. We present measurements of the threshold for structural change in Corning 0211 glass as well as a study of the morphology of the structures produced by single and multiple laser pulses. At a high repetition rate, multiple pulses produce a structural change dominated by cumulative heating of the material by successive laser pulses. Using this cumulative heating effect, we write single-mode optical waveguides inside bulk glass, using only a laser oscillator.

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

We demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures (the external capsule (EC) and hippocampus) within an intact mouse brain using three-photon fluorescence microscopy at the new spectral window of 1700 nm.

Laser-induced breakdown and damage in bulk transparent materials induced by tightly focused femtosecond laser pulses

Laser-induced breakdown and damage to transparent materials has remained an active area of research for four decades. In this paper we review the basic mechanisms that lead to laser-induced breakdown and damage and present a summary of some open questions in the field. We present a method for measuring the threshold intensity required to produce breakdown and damage in the bulk, as opposed to on the surface, of the material. Using this technique, we measure the material band-gap and laser-wavelength dependence of the threshold intensity for bulk damage using femtosecond laser pulses. Based on these thresholds, we determine the relative role of different nonlinear ionization mechanisms for different laser and material parameters.

Deep tissue multiphoton microscopy using longer wavelength excitation

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 mum are performed.

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

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.

Penetrating arterioles are a bottleneck in the perfusion of neocortex

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.

Dynamics of femtosecond laser-induced breakdown in water from femtoseconds to microseconds

Using time-resolved imaging and scattering techniques, we directly and indirectly monitor the breakdown dynamics induced in water by femtosecond laser pulses over eight orders of magnitude in time. We resolve, for the first time, the picosecond plasma dynamics and observe a 20 ps delay before the laser-produced plasma expands. We attribute this delay to the electron-ion energy transfer time.

All-Optical Histology Using Ultrashort Laser Pulses

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 microJ 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 100 microm 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.

Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain

The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to image both blood flow and the activity of individual cells below the surface of the brain. Here we provide a technical guide to imaging cerebral blood flow in rodents. We describe in detail the surgical procedures required to generate cranial windows for optical access to the cortex of both rats and mice and the use of two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We further provide examples on how these techniques can be applied to the study of local blood flow regulation and vascular pathologies such as small-scale stroke.

Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration

Inhibiting endothelial cell contractility reverses the deleterious effects of age-related matrix stiffening on normal cell function, which could help prevent the development of atherosclerosis. Rock Your Heart Out According to novelist Thomas Bailey Aldrich, “To keep the heart unwrinkled, to be hopeful, kindly, cheerful, reverent, is to triumph over old age” (from Ponkapoag Papers). Unfortunately, despite a positive attitude, aging is accompanied by several changes of heart, at least at the cellular level. One age-related “wrinkle” is stiffening of the extracellular matrix that lines the blood vessels, a change that has been linked to atherosclerosis; yet, the cellular and mechanical features that couple the two conditions have remained elusive. Now, using a clever combination of biomaterials, cells, aortas, and mice, Huynh and colleagues have demystified the correlation between aging and atherosclerosis, showing that cell contractility is at the heart of it all. The authors first developed an in vitro system that mimicked the basic structures of both young and old blood vessels. Synthetic hydrogel matrices of varying stiffnesses were seeded with bovine aortic endothelial cells. By administering a solution of fluorescently labeled molecules to the cell-gel system and watching how the dye moved across the cell layer, Huynh et al. determined that permeability increased as a function of matrix stiffness, suggesting that age alone was a disruptive factor. These results were confirmed ex vivo by performing atomic force microscopy with decellularized thoracic aortas from both young (~10 weeks) and old (~92 weeks) mice. In both of these systems, the enhanced vessel permeability resulted from an increase in the distance—or junction—between neighboring cells. This increase in the so-called gap junction width also permitted the passage of leukocytes through the endothelial cell monolayer; along with leaky vasculature, cellular transmigration is a hallmark of atherosclerosis progression. Because the Rho signaling pathway is linked to the cellular cytoskeleton and, in turn, contractility, Huynh et al. hypothesized that they could reverse the effects of age-related intimal stiffening by inhibiting Rho-associated kinase (ROCK). By administering a pharmacological ROCK inhibitor (Y-27632) to their in vitro setup and to old mice, the authors showed that gap junction widths and endothelial cellular forces decreased. In vitro, the inhibitor also prevented leukocyte transmigration. These observations suggest that directly interfering with Rho signaling is a viable treatment option for age-related atherosclerosis. And because inhibitors of Rho signaling, such as fasudil, are already available in the clinic, one might say that physicians and researchers are ready to rock. Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression.