Biomedical subjects
Publications and source records attributed to Stephen P Nighswander-Rempel.
This article demonstrates that a commonly-made assumption in quantum yield calculations may produce errors of up to 25% in extreme cases and can be corrected by a simple modification to the analysis.
The quantum yield of synthetic eumelanin is known to be extremely low and it has recently been reported to be dependent on excitation wavelength. In this paper, we present quantum yield as a function of excitation wavelength between 250 and 500 nm, showing it to be a factor of 4 higher at 250 nm than at 500 nm. In addition, we present a definitive map of the steady-state fluorescence as a function of excitation and emission wavelengths, and significantly, a three-dimensional map of the "specific quantum yield": the fraction of photons absorbed at each wavelength that are subsequently radiated at each emission wavelength. This map contains clear features, which we attribute to certain structural models, and shows that radiative emission and specific quantum yield are negligible at emission wavelengths outside the range of 585 and 385 nm (2.2 and 3.2 eV), regardless of excitation wavelength. This information is important in the context of understanding melanin biofunctionality, and the quantum molecular biophysics therein.
Previously reported excitation spectra for eumelanin are sparse and inconsistent. Moreover, these studies have failed to account for probe beam attenuation and emission reabsorption within the samples, making them qualitative at best. We report for the first time quantitative excitation spectra for synthetic eumelanin, acquired for a range of solution concentrations and emission wavelengths. Our data indicate that probe beam attenuation and emission reabsorption significantly affect the spectra even in low-concentration eumelanin solutions and that previously published data do not reflect the true excitation profile. We apply a correction procedure (previously applied to emission spectra) to account for these effects. Application of this procedure reconstructs the expected relationship of signal intensity with concentration, and the normalized spectra show a similarity in form to the absorption profiles. These spectra reveal valuable information regarding the photophysics and photochemistry of eumelanin. Most notably, an excitation peak at 365 nm (3.40 eV), whose position is independent of emission wavelength, is possibly attributable to a 5,6-dihydroxyindole-2-carboxylic acid (DHICA) component singly linked to a polymeric structure.
Near-infrared (NIR) spectroscopic imaging is emerging as a unique tool for intra-operative assessment of myocardial oxygenation, but quantitative interpretation of the images is not straightforward. One confounding factor specific to muscle tissue (both skeletal and cardiac) is that the visible/NIR absorbance spectrum of myoglobin (Mb), an intracellular O(2) storage protein, is virtually identical to that of hemoglobin (Hb). As a consequence, the relative contributions of Mb and Hb to the NIR spectra measured in vivo for blood perfused muscle tissue cannot be determined from the measured spectra alone. To estimate the relative contributions of Mb and Hb to NIR spectra and spectroscopic images, isolated pig hearts were perfused first with a Hb-free blood substitute (Krebs-Henseleit buffer; KHB) and then with a 50/50 KHB/blood mixture, with spectroscopic images acquired at each step. Tissue Mb levels were estimated directly from the measurements during KHB perfusion, and total (Mb+Hb) levels were estimated from the images acquired during 50/50 blood/KHB perfusion. Myoglobin accounted for 63 +/- 11% of the total heme content during perfusion with the 50/50 mixture (implying that Mb would contribute 46% of the combined (Mb+Hb) NIR profile during whole blood perfusion), confirming that Mb contributes substantially to near-infrared absorbance spectra of blood perfused cardiac tissue.
Near-infrared fibre-optic single point spectroscopy has been widely exploited to provide information regarding blood volume and oxygenation in vivo, but it does not provide any information on regional differences in perfusion. We have combined the chemical sensitivity of spectroscopy with the spatial sensitivity of imaging to generate maps of regional cardiac oxygenation. Spectroscopic images were acquired for isolated, arrested, blood-perfused porcine hearts (n=4) over the wavelength range 650 and 1050 nm. Spectroscopic images were acquired during normal perfusion, regional ischemia (occlusion of left anterior descending artery) global ischemia, and reperfusion. Hemoglobin (Hb) and myoglobin (Mb) content and oxygenation were determined by reconstructing the tissue spectra measured at each pixel as weighted sums of water, oxy- and deoxy-Hb (and -Mb) absorptivity spectra. The spectroscopic images acquired during regional ischemia clearly revealed increased deoxy-(Hb+Mb) levels and decreased oxy-(Hb+Mb) levels in the ischaemic regions relative to the normally-perfused regions. Global ischemia produced a dramatic decrease in oxy-(Hb+Mb) levels and a moderate increase in deoxy-(Hb+Mb). These images confirm that blood oxygenation can be mapped in cardiac tissue by near-infrared spectroscopic imaging.
One hallmark of cell death resulting from prolonged ischemia is cell membrane disruption. We apply optical spectroscopy to gauge membrane disruption in isolated rat hearts by monitoring (1) the washout of myoglobin (Mb) and (2) the accumulation of an exogenous contrast agent in permeabilized cells. The contrast agent, a neodymium (Nd) chelate, has several absorptions in the visible and near-IR, and when present in the perfusate, it cannot penetrate cellular membranes. When membrane integrity is disrupted, however, it is expected to accumulate within the intracellular space; moreover, cellular Mb is expected to wash out. To test this hypothesis, rat hearts (n=12) are perfused with Krebs-Henseleit buffer (KHB), followed by perfusion with KHB in which a 5 mM Nd-DTPA solution is present. Membrane damage is then induced by infusion of digitonin into the Nd-KHB perfusate to provide a digitonin concentration of 2.5, 5, or 10 microg/mL. After 30 min of infusion, Mb levels fall to 46+/-14% of baseline levels and Nd-DTPA rises to 161+/-19% of predigitonin levels. No apparent dependence of total membrane disruption on digitonin concentration over the concentration range studied is found, although higher concentrations do lead to more rapid membrane disruption.
Quantitative analysis of blood oxygen saturation using near-IR spectroscopy is made difficult by uncertainties in both the absolute value and the wavelength dependence of the optical path length. We introduce a novel means of assessing the wavelength dependence of path length, exploiting the relative intensities of several absorptions exhibited by an exogenous contrast agent (neodymium). Combined with a previously described method that exploits endogenous water absorptions, the described technique estimates the absolute path length at several wavelengths throughout the visible/near-IR range of interest. Isolated rat hearts (n = 11) are perfused separately with Krebs-Henseleit buffer (KHB) and a KHB solution to which neodymium had been added, and visible/near-IR spectra are acquired using an optical probe made up of emission and collection fibers in concentric rings of diameters 1 and 3 mm, respectively. Relative optical path lengths at 520, 580, 679, 740, 800, 870, and 975 nm are 0.41+/-0.13, 0.49+/-0.21, 0.90+/-0.09, 0.94+/-0.01, 1.00, 0.84+/-0.01, and 0.78+/-0.08, respectively. The absolute path length at 975 nm is estimated to be 3.8+/-0.6 mm, based on the intensity of the water absorptions and the known tissue water concentration. These results are strictly valid only for the experimental geometry applied here.
Near-infrared spectroscopic imaging (NIRSI) is useful to assess cardiac tissue oxygenation in arrested and beating hearts, and it shows potential as an intraoperative gauge of the effectiveness of bypass grafting. The purpose of this study was to determine whether NIRSI can reliably differentiate among a range of cardiac oxygenation states, using ischemia and hypoxia models independently. An ischemia-reperfusion model was applied to isolated, beating, blood-perfused porcine hearts, in which the left anterior descending (LAD) artery was cannulated. LAD flow was decreased stepwise to approximately 50, 20, and 0% of normal flow and was completely restored between ischemic episodes. Upon completion of the ischemia-reperfusion protocol, the hearts were further subjected to periods of increasingly severe global hypoxia. Regional oxy- and deoxy-hemoglobin (myoglobin) levels were derived from spectroscopic images (650 to 1050 nm) acquired at each step. Oxygenation maps vividly highlighted the area at risk for all degrees of ischemia. Oxygenation values differed significantly for different LAD flow rates, regardless of whether intermediate reperfusion was applied, and oxygenation values during progressive hypoxia correlated well with blood oxygen saturation. These results suggest that NIRSI is well suited, not only to identify ischemic or hypoxic regions of cardiac tissue, but also to assess the severity of deoxygenation.