Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within  d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibro- blast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P < 0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained con- trols, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and corre- lated with direction (P < 0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.

Background: The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions. Methodology/Principal Findings: We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 mm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters. Conclusions/Significance: To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies.

The direct thrombin inhibitor dabigatran etexilate (DE) may constitute a future replacement of vitamin K antagonists for long-term anticoagulation. Whereas warfarin pretreatment is associated with greater hematoma expansion after intracerebral hemorrhage (ICH), it remains unclear what effect direct thrombin inhibitors would have. Using different experimental models of ICH, this study compared hematoma volume among DE-treated mice, warfarin-treated mice, and controls. Methods and Results—CD-1 mice were fed with DE or warfarin. Sham-treated mice served as controls. At the time point of ICH induction, DE mice revealed an increased activated partial thromboplastin time compared with controls (mean+/-SD 46.1+/-5.0 versus 18.0+/-1.5 seconds; P=0.022), whereas warfarin pretreatment resulted in a prothrombin time prolongation (51.4+/-17.9 versus 10.4+/-0.3 seconds; P<0.001). Twenty-four hours after collagenase-induced ICH formation, hematoma volume was 3.8+/-2.9 µL in controls, 4.8+/-2.7 µL in DE mice, and 14.5+/-11.8 µL in warfarin mice (n=16; Welch ANOVA between-group differences P􏰁0.007; posthoc analysis with the Dunnett method: DE versus controls, P=0.899; warfarin versus controls, P<0.001; DE versus warfarin, P=0.001). In addition, a model of laser-induced cerebral microhemorrhage was applied, and the distances that red blood cells and blood plasma were pushed into the brain were quantified. Warfarin mice showed enlarged red blood cell and blood plasma diameters compared to controls, but no difference was found between DE mice and controls. Conclusions—In contrast with warfarin, pretreatment with DE did not increase hematoma volume in 2 different experimental models of ICH. In terms of safety, this observation may represent a potential advantage of anticoagulation with DE over warfarin.

In vivo imaging allows detailed studies of cerebral blood flow and brain metabolism in both normal and pathological states. Since the 1980s, several methods to measure tissue perfusion in the brain have been introduced. Magnetic resonance imaging (MRI) tech- niques such as blood oxygen level dependent MRI (BOLD MRI) (Sorensen et al, 1995) and arterial spin labeling (Buxton and Frank 1997) as well as micro- positron emission tomography (Heiss et al, 1994) have enabled noninvasive chronic imaging of both regional blood flow changes and absolute perfusion in both humans and animal models. These methods, however, suffer from poor spatial resolution and are mainly useful when studying regional blood flow in the brain. Intrinsic optical imaging (Ts’o et al, 1990) enables higher spatial resolution mapping of changes in blood volume. Laser Doppler spectroscopy (Eyre et al, 1988) enables measurement of relative changes in blood flow speeds averaged over B1-mm2 cortical areas, while laser speckle contrast imaging (Boas and Dunn, 2010) enables these relative blood flow changes to be imaged with a spatial resolution of < 100 mm. None of these optical techniques, however, can accurately resolve changes in flow in individual microvessels or determine the absolute perfusion of cortical tissue. When absolute speed measurement of individual vessels is required, two-photon excited fluorescence (2PEF) microscopy has been the tool of choice (Schaffer et al, 2006). It is, however, limited to measurement of flow speed in vessels that are oriented parallel to the imaging plane, and measure- ments need to be made one vessel at a time, which is a time-consuming process. Currently lacking is an imaging technique that enables absolute blood flow speed measurements in multiple individual vessels at once. In this issue of JCBFM, Srinivasan et al (2011) introduce the use of Doppler optical coherence tomography (DOCT) (Chen et al, 1997) to fill this gap.

Non-reciprocal optical phenomena — effects that depend on the direction of light propagation —are rare. Researchers have now observed non-reciprocal material modification when moving a beam of ultrashort light pulses through a lithium niobate crystal.

Porphyrin molecules have a highly conjugated cyclic structure and are theorized to have unusually large two-photon absorptivities (σTPA), i.e., σTPA ~ 10^2 GM. The authors tested this claim. Ultrafast two-photon absorption (TPA) spectroscopy was performed on solutions of hemoglobin, which contains a naturally occurring metaloporphyrin. They used a pump-probe technique to directly detect the change in transmission induced by TPA over the wavelength range of λ0=780–880 nm. As controls, they measured the TPA of the dyes rhodamine 6G and B; their measurements both verify and extend previously reported values. In new results, hemoglobin was found to have a peak two-photon absorptivity of σTPA~150 GM at λ0=825 nm, near a resonance of the Soret band. This value supports theoretical expectations. They also found a significant difference in the TPA of carboxyhemoglobin versus oxyhemoglobin, e.g., σTPA=61 GM versus σTPA=18 GM, respectively, at λ0=850 nm, which shows that the ligand affects the electronic states involved in TPA.

Femtosecond laser drilling is used to produce a variable-pressure fiber gas cell. Tightly focused laser pulses are used to produce micrometer-diameter radial channels in a hollow-core photonic band-gap fiber (HC-PBGF), and through these microchannels the core of the fiber is filled with a gas. The fiber cell is formed by fusion splicing and sealing the ends of the HC-PBGF to standard step-index fiber. As a demonstration, acetylene is introduced into an evacuated fiber at multiple backing pressures and spectra are measured.

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.