Application of a Novel Murine Ear Vein Model to Evaluate the Effects of a Vascular Radioprotectant on Radiation-Induced Vascular Permeability and Leukocyte Adhesion.

Vascular injury after radiation exposure contributes to multiple types of tissue injury through a cascade of events. Some of the earliest consequences of radiation damage include increased vascular permeability and promotion of inflammation, which is partially manifested by increased leukocyte-endothelial (L/E) interactions. We describe herein a novel intravital imaging method to evaluate L/E interactions, as a function of shear stress, and vascular permeability at multiple time points after local irradiation to the ear. This model permitted analysis of quiescent vasculature that was not perturbed by any surgical manipulation prior to imaging. To evaluate the effects of radiation on vascular integrity, fluorescent dextran was injected intravenously and its extravasation in the extravascular space surrounding the ear vasculature was measured at days 3 and 7 after 6 Gy irradiation. The vascular permeability rate increased approximately twofold at both days 3 and 7 postirradiation ( P < 0.05). Leukocyte rolling, which is indicative of L/E interactions, was significantly increased in mice at 24 h postirradiation compared to that of nonirradiated mice. To assess our model, as a means for assessing vascular radioprotectants, we treated additional cohorts of mice with a thrombopoietin mimetic, TPOm (RWJ-800088). In addition to stimulating platelet formation, thrombopoietin can protect vasculature after several forms of injury. Thus, we hypothesized that TPOm would reduce vascular permeability and L/E adhesion after localized irradiation to the ear vasculature of mice. If TPOm reduced these consequences of radiation, it would validate the utility of our intravital imaging method. TPOm reduced radiation-induced vascular leakage to control levels at day 7. Furthermore, L/E cell interactions were also reduced in irradiated mice treated with TPOm, compared with mice receiving irradiation alone, particularly at high shear stress ( P = 0.03, Kruskal-Wallis). We conclude that the ear model is useful for monitoring quiescent normal tissue vascular injury after radiation exposure. Furthermore, the application of TPOm, for preventing early inflammatory response created by damage to vascular endothelium, suggests that this drug may prove useful in reducing toxicities from radiotherapy, which damage microvasculature that critically important to tissue function.

Tunable and amplified Raman gold nanoprobes for effective tracking (TARGET): in vivo sensing and imaging.

We describe the development of a highly tunable, physiologically stable, and ultra-bright Raman probe, named as TARGET (Tunable and Amplified Raman Gold Nanoprobes for Effective Tracking), for in vitro and in vivo surface-enhanced Raman scattering (SERS) applications. The TARGET structure consists of a gold core inside a larger gold shell with a tunable interstitial gap similar to a "nanorattle" structure. The combination of galvanic replacement and the seed mediated growth method was employed to load Raman reporter molecules and subsequently close the pores to prevent leaking and degradation of reporters under physiologically extreme conditions. Precise tuning of the core-shell gap width, core size, and shell thickness allows us to modulate the plasmonic effect and achieve a maximum electric-field (E-field) intensity. The interstitial gap of TARGET nanoprobes can be designed to exhibit a plasmon absorption band at 785 nm, which is in resonance with the dye absorption maximum and lies in the "tissue optical window", resulting in ultra-bright SERS signals for in vivo studies. The results of in vivo measurements of TARGETs in laboratory mice illustrated the usefulness of these nanoprobes for medical sensing and imaging.

Snap-shot multispectral imaging of vascular dynamics in a mouse window-chamber model.

Understanding tumor vascular dynamics through parameters such as blood flow and oxygenation can yield insight into tumor biology and therapeutic response. Hyperspectral microscopy enables optical detection of hemoglobin saturation or blood velocity by either acquiring multiple images that are spectrally distinct or by rapid acquisition at a single wavelength over time. However, the serial acquisition of spectral images over time prevents the ability to monitor rapid changes in vascular dynamics and cannot monitor concurrent changes in oxygenation and flow rate. Here, we introduce snap shot-multispectral imaging (SS-MSI) for use in imaging the microvasculature in mouse dorsal-window chambers. By spatially multiplexing spectral information into a single-image capture, simultaneous acquisition of dynamic hemoglobin saturation and blood flow over time is achieved down to the capillary level and provides an improved optical tool for monitoring rapid in vivo vascular dynamics.

Oxygen Sensing Difluoroboron Dinaphthoylmethane Polylactide.

Dual emissive luminescence properties of solid-state difluoroboron ß-diketonate-poly(lactic acid) (BF2bdk-PLA) materials have been utilized as biological oxygen sensors. Dyes with red-shifted absorption and emission are important for multiplexing and in vivo imaging, thus hydroxyl-functionalized dinaphthoylmethane initiators and dye-PLA conjugates BF2dnm(X)PLA (X = H, Br, I) with extended conjugation were synthesized. The luminescent materials show red-shifted absorbance (~435 nm) and fluorescence tunability by molecular weight. Fluorescence colors range from yellow (~530 nm) in 10 - 12 kDa polymers to green (~490 nm) in 20 - 30 kDa polymers. Room-temperature phosphorescence (RTP) and thermally activated delayed fluorescence (TADF) are present under a nitrogen atmosphere. For the iodine-substituted derivative, BF2dnm(I)PLA, clearly distinguishable fluorescence (green) and phosphorescence (orange) peaks are present, making it ideal for ratiometric oxygen-sensing and imaging. Bromide and hydrogen analogues with weaker relative phosphorescence intensities and longer phosphorescence lifetimes can be used as highly sensitive, concentration independent, lifetime-based oxygen sensors or for gated emission detection. BF2dnm(I)PLA nanoparticles were taken up by T41 mouse mammary cells and successfully demonstrated differences in vitro ratiometric measurement of oxygen.

Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion.

Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ~42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., "PIPA") restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ~120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo.

Automated non-rigid registration and mosaicing for robust imaging of distinct retinal capillary beds using speckle variance optical coherence tomography.

Variance processing methods in Fourier domain optical coherence tomography (FD-OCT) have enabled depth-resolved visualization of the capillary beds in the retina due to the development of imaging systems capable of acquiring A-scan data in the 100 kHz regime. However, acquisition of volumetric variance data sets still requires several seconds of acquisition time, even with high speed systems. Movement of the subject during this time span is sufficient to corrupt visualization of the vasculature. We demonstrate a method to eliminate motion artifacts in speckle variance FD-OCT images of the retinal vasculature by creating a composite image from multiple volumes of data acquired sequentially. Slight changes in the orientation of the subject's eye relative to the optical system between acquired volumes may result in non-rigid warping of the image. Thus, we use a B-spline based free form deformation method to automatically register variance images from multiple volumes to obtain a motion-free composite image of the retinal vessels. We extend this technique to automatically mosaic individual vascular images into a widefield image of the retinal vasculature.

Depth-Encoded Spectral Domain Phase Microscopy for Simultaneous Multi-Site Nanoscale Optical Measurements.

Spectral domain phase microscopy (SDPM) is an extension of spectral domain optical coherence tomography (SDOCT) that exploits the extraordinary phase stability of spectrometer-based systems with common-path geometry to resolve sub-wavelength displacements within a sample volume. This technique has been implemented for high resolution axial displacement and velocity measurements in biological samples, but since axial displacement information is acquired serially along the lateral dimension, it has been unable to measure fast temporal dynamics in extended samples. Depth-Encoded SDPM (DESDPM) uses multiple sample arms with unevenly spaced common path reference reflectors to multiplex independent SDPM signals from separate lateral positions on a sample simultaneously using a single interferometer, thereby reducing the time required to detect unique optical events to the integration period of the detector. Here, we introduce DESDPM and demonstrate the ability to acquire useful phase data concurrently at two laterally separated locations in a phantom sample as well as a biological preparation of spontaneously beating chick cardiomyocytes. DESDPM may be a useful tool for imaging fast cellular phenomena such as nervous conduction velocity or contractile motion.

Doppler velocity detection limitations in spectrometer-based versus swept-source optical coherence tomography.

Recent advances in Doppler techniques have enabled high sensitivity imaging of biological flow to measure blood velocities and vascular perfusion. Here we compare spectrometer-based and wavelength-swept Doppler OCT implementations theoretically and experimentally, characterizing the lower and upper observable velocity limits in each configuration. We specifically characterize the washout limit for Doppler OCT, the velocity at which signal degradation results in loss of flow information, which is valid for both quantitative and qualitative flow imaging techniques. We also clearly differentiate the washout effect from the separate phenomenon of phase wrapping. We demonstrate that the maximum detectable Doppler velocity is determined by the fringe washout limit and not phase wrapping. Both theory and experimental results from phantom flow data and retinal blood flow data demonstrate the superiority of the swept-source technique for imaging vessels with high flow rates.

Synthetic wavelength based phase unwrapping in spectral domain optical coherence tomography.

Phase sensing implementations of spectral domain optical coherence tomography (SDOCT) have demonstrated the ability to measure nanometer-scale temporal and spatial profiles of samples. However, the phase information suffers from a 2pi ambiguity that limits observations of larger sample displacements to lengths less than half the source center wavelength. We introduce a synthetic wavelength phase unwrapping technique in SDOCT that uses spectral windowing and corrects the 2pi ambiguity, providing accurate measurements of sample motion with information gained from standard SDOCT processing. We demonstrate this technique by using a common path implementation of SDOCT and correctly measure phase profiles from a phantom phase object and human epithelial cheek cells which produce multiple wrapping artifacts. Using a synthetic wavelength for phase unwrapping could prove useful in Doppler or other phase based implementations of OCT.