Evidence of a heterogeneous tissue oxygenation: renal ischemia/reperfusion injury in a large animal model.

Renal ischemia that occurs intraoperatively during procedures requiring clamping of the renal artery (such as renal procurement for transplantation and partial nephrectomy for renal cancer) is known to have a significant impact on the viability of that kidney. To better understand the dynamics of intraoperative renal ischemia and recovery of renal oxygenation during reperfusion, a visible reflectance imaging system (VRIS) was developed to measure renal oxygenation during renal artery clamping in both cooled and warm porcine kidneys. For all kidneys, normothermic and hypothermic, visible reflectance imaging demonstrated a spatially distinct decrease in the relative oxy-hemoglobin concentration (%HbO2) of the superior pole of the kidney compared to the middle or inferior pole. Mean relative oxy-hemoglobin concentrations decrease more significantly during ischemia for normothermic kidneys compared to hypothermic kidneys. VRIS may be broadly applicable to provide an indicator of organ ischemia during open and laparoscopic procedures.

Vibrational spectroscopy of biomembranes.

Vibrational spectroscopy, commonly associated with IR absorption and Raman scattering, has provided a powerful approach for investigating interactions between biomolecules that make up cellular membranes. Because the IR and Raman signals arise from the intrinsic properties of these molecules, vibrational spectroscopy probes the delicate interactions that regulate biomembranes with minimal perturbation. Numerous innovative measurements, including nonlinear optical processes and confined bilayer assemblies, have provided new insights into membrane behavior. In this review, we highlight the use of vibrational spectroscopy to study lipid-lipid interactions. We also examine recent work in which vibrational measurements have been used to investigate the incorporation of peptides and proteins into lipid bilayers, and we discuss the interactions of small molecules and drugs with membrane structures. Emerging techniques and measurements on intact cellular membranes provide a prospective on the future of vibrational spectroscopic studies of biomembranes.

Visual enhancement of laparoscopic partial nephrectomy with 3-charge coupled device camera: assessing intraoperative tissue perfusion and vascular anatomy by visible hemoglobin spectral response.

PURPOSE: We report the novel use of 3-charge coupled device camera technology to infer tissue oxygenation. The technique can aid surgeons to reliably differentiate vascular structures and noninvasively assess laparoscopic intraoperative changes in renal tissue perfusion during and after warm ischemia. MATERIALS AND METHODS: We analyzed select digital video images from 10 laparoscopic partial nephrectomies for their individual 3-charge coupled device response. We enhanced surgical images by subtracting the red charge coupled device response from the blue response and overlaying the calculated image on the original image. Mean intensity values for regions of interest were compared and used to differentiate arterial and venous vasculature, and ischemic and nonischemic renal parenchyma. RESULTS: The 3-charge coupled device enhanced images clearly delineated the vessels in all cases. Arteries were indicated by an intense red color while veins were shown in blue. Differences in mean region of interest intensity values for arteries and veins were statistically significant (p >0.0001). Three-charge coupled device analysis of pre-clamp and post-clamp renal images revealed visible, dramatic color enhancement for ischemic vs nonischemic kidneys. Differences in the mean region of interest intensity values were also significant (p <0.05). CONCLUSIONS: We present a simple use of conventional 3-charge coupled device camera technology in a way that may provide urological surgeons with the ability to reliably distinguish vascular structures during hilar dissection, and detect and monitor changes in renal tissue perfusion during and after warm ischemia.

Lipid vesicle aggregation induced by cooling.

Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 degrees C to 5 degrees C) and about 2 micron vesicles of DSPC (from 20 degrees C to 15 degrees C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 degrees C to 5 degrees C failed to increase aggregation within 24 hours at Mg(++)/DPPS ratios that begin to initiate aggregation and fusion.

Advantages and artifacts of higher order modes in nanoparticle-enhanced backscattering Raman imaging.

In order to facilitate nanoparticle-enhanced Raman imaging of complicated biological specimens, we have examined the use of higher order modes with radial and azimuthal polarizations focused onto a Au nanoparticle atomic force microscope (AFM) tip utilizing a backscattering reflection configuration. When comparing the Raman intensity profiles with the observed sample topography, the radial-polarized configuration demonstrates enhanced spatial resolution. This enhanced resolution results from the direction of the induced electron oscillation in the metal nanoparticle oriented by the electromagnetic field at the laser focus. The electric field component along the direction of laser propagation, attendant to the radial polarization, creates an enhanced field along the z-axis and normal to the sample. Substantial enhancement is observed utilizing an intermediate numerical aperture objective (NA = 0.7), necessary for backscattering measurements. The azimuthal polarization, similar to linear polarization, results in an enhanced field predominantly parallel to the sample, resulting in imaging artifacts. The Raman intensity profiles observed as the exciting laser polarization is switched between either a radially polarized or an azimuthally polarized state illustrate these imaging artifacts. Because azimuthal polarization arises readily from changes in the incident polarization onto the mode converter, the results presented here aid in identifying such artifacts when analyzing nanoparticle-enhanced Raman spectroscopic images. Due to the power law decay of the enhanced field, an enhancement orientation normal to the sample enables contrast between structures smaller than the tip dimensions as the apex of the nanoparticle tip, where the enhancement is strongest, passes over the sample. These effects are demonstrated using both carbon nanotube and fixed biological samples.

Infrared spectroscopic imaging of latent fingerprints and associated forensic evidence.

Fingerprints reflecting a specific chemical history, such as exposure to explosives, are clearly distinguished from overlapping, and interfering latent fingerprints using infrared spectroscopic imaging techniques and multivariate analysis.

Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

Non-invasive detection of superimposed latent fingerprints and inter-ridge trace evidence by infrared spectroscopic imaging.

Current latent print and trace evidence collecting technologies are usually invasive and can be destructive to the original deposits. We describe a non-invasive vibrational spectroscopic approach that yields latent fingerprints that are overlaid on top of one another or that may contain trace evidence that needs to be distinguished from the print. Because of the variation in the chemical composition distribution within the fingerprint, we demonstrate that linear unmixing applied to the spectral content of the data can be used to provide images that reveal superimposed fingerprints. In addition, we demonstrate that the chemical composition of the trace evidence located in the region of the print can potentially be identified by its infrared spectrum. Thus, trace evidence found at a crime scene that previously could not be directly related to an individual, now has the potential to be directly related by its presence in the individual-identifying fingerprints.

Tip-enhanced Raman spectroscopy and imaging: an apical illumination geometry.

Results are presented illustrating the use of tip-enhanced Raman spectroscopy (TERS) and imaging in a top-illumination geometry. A radially polarized beam is used to generate an electric field component in the direction of beam propagation, normal to the surface, resulting in a 5x increased enhancement compared to a linearly polarized beam. This multiplicative enhancement facilitates a discrimination of the near-field signal from the far-field Raman background. The top illumination configuration facilitates the application of TERS for investigating molecules on a variety of surfaces, such as Au, glass, and Si. The near-field Raman spectra of Si(100), rhodamine B, brilliant cresyl blue, and single wall carbon nanotubes are presented. Sufficient enhancement is obtained to permit a sub-diffraction-limited resolution Raman imaging of the surface distribution of large bundles of carbon nanotubes of various diameters.

Pharmacodynamic assessment of histone deacetylase inhibitors: infrared vibrational spectroscopic imaging of protein acetylation.

Infrared spectroscopy identifies molecules by detection of vibrational patterns characteristic of molecular bonds. We apply this approach to measure protein acetylation after treatment with histone deacetylase inhibitors. The anticancer activity of histone deacetylase inhibitors (HDACi) is ascribed to the hyperacetylation of both core nucleosomal histones and nonhistone proteins critical to the maintenance of the malignant phenotype (Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Curr. Opin. Oncol. 2001, 13, 477-483; Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.; Simeoni, S.; Ragno, R. Med. Res. Rev. 2005, 25, 261-309). After incubation of the peripheral blood mononuclear cells (PBMCs) in vitro with the HDACi SNDX-275, a benzamide drug derivative, vibrational spectral changes in the methyl and methylene stretching mode regions, which reflect concentration-dependent increases in protein acetylation, were detected and quantified. We applied these metrics, based upon spectral differences, to peripheral blood mononuclear cells from patients treated in vivo with this agent. The data demonstrate a new approach to a sensitive assessment of global molecular modifications that is independent of antibodies, requires minimal cell processing, and is easily adapted to high-throughput screening.