Stephen P. Nighswander-Rempel

Relative Contributions of Hemoglobin and Myoglobin to Near-Infrared Spectroscopic Images of Cardiac Tissue:

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 O2 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.

Effect of dimerization on vibrational spectra of eumelanin precursors.

We have synthesized a compound ideally suited to the study of structure‐function relationships in eumelanin synthesis. N‐methyl‐5‐hydroxy‐6‐methoxy‐indole (MHMI) has key functional groups strategically placed on the indole framework to hinder binding in the 2, 5, 6 and 7 positions. Thus, the dimer bound exclusively in the 4‐4′ positions was isolated and characterized. In order to study the difference in vibrational structure between the MHMI monomer and dimer, Raman spectra were acquired of both compounds, as well as indole, indole‐2‐carboxylic acid and 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA). Peaks were assigned to particular vibrational modes using B3LYP density functional theory calculations, and experimental and theoretical spectra displayed good agreement. Addition of functional groups to either benzene or pyrrole rings in the indole framework impacted vibrational spectra attributed to vibrations in either ring, and in some cases, peaks appearing unchanged between two compounds corresponded to different contributing vibrations. Dimerization resulted in an expected increase in the number of vibrational modes, but not a significant increase in the number of apparent peaks, as several modes frequently contributed to an individual observed peak. Comparison of spectral features of the monomer and dimer provides insight into eumelanin photochemistry, but final conclusions depend on the planarity of oligomeric structure in vivo.

Assessment of optical path length in tissue using neodymium and water absorptions for application to near-infrared spectroscopy

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.

Time‐Resolved and Steady‐State Fluorescence Spectroscopy of Eumelanin and Indolic Polymers

Eumelanin plays a variety of important physiological roles in human skin. However, its structure and fundamental properties still remain poorly understood. Although the absorbance of eumelanin is broad and reveals little about its structure, a variety of techniques have revealed the presence of a disordered array of chromophores within the melanin compound. In order to examine the fluorescence decay dynamics of these chromophores, time‐resolved spectroscopy was applied to solutions of synthetic eumelanin and a melanin‐like polymer of N‐methyl,5‐hydroxy,6‐methoxyindole (N‐Me‐5H6MI). Solutions were excited with 80 fs laser pulses at 355, 370, 390 and 400 nm, and decay time courses were acquired at 20 nm intervals between 400 and 600 nm for each excitation wavelength. Decay profiles for both eumelanin and the polymer exhibited a characteristic multiexponential behavior with decay times between 0.5 and 15 ns, although steady‐state spectra for the polymer exhibited only two peaks. The long‐decay component in the polymer showed a significant decrease in both amplitude (30–5%) and decay time (14–6 ns) with increasing emission wavelength. In contrast, the amplitude and decay time in melanin increased slightly (10–15% and 7–10 ns, respectively) from 400 to 520 nm emission, at which point they leveled off. These trends were consistent for all excitation wavelengths. These results suggest that the multiexponential behavior of melanin fluorescence is characteristic of each oligomer within the eumelanin compound, and is consistent with the assertion that the diversity of constituents within eumelanin provides it with a robustness in spectral properties.

Quantum yield calculations for strongly absorbing chromophores

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.

Detection of myocardial cell damage in isolated rat hearts with near-infrared spectroscopy

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.

Solvochromic Effects in Model Eumelanin Compounds

We have created an indolic compound which is ideally suited to the study of the relationship between structure and function in eumelanin formation. N‐methyl‐5‐hydroxy‐6‐methoxyindole (MHMI) is stable in solid and liquid states, highly soluble in a variety of solvents and forms a dimer only through the 4‐4′ positions. The limited binding possibilities are due to functional groups strategically placed to inhibit chemical interactions through the 2 and 7 positions. It forms a crystal structure with a remarkable packing arrangement, with four monomers grouped in parallel pairs spaced 3.5 Å apart within each unit cell. Optical spectra reveal a multi‐peaked absorbance profile similar to 5,6‐dihydroxyindole (DHI) and N‐acetyl‐tryptophanamide (NATA), and strong fluorescence emission with radiative quantum yields of 29% and 33% in benzene and acetonitrile, respectively. The quantum yield is similar to that of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and shows that solvent aromaticity by itself does not affect the yield. Solution in chloroform results in an almost complete quenching of the fluorescence but an increase in emission is observed with photoactivation. Crystallographic results shown here suggest new structural possibilities for eumelanin and the controlled binding possibilities make this an excellent model for monitoring changes in function with increasing oligomer size in eumelanin formation.

Neodymium and Water Absorptions Can Reveal the Wavelength Dependence of Optical Pathlength for in vivo near Infrared Spectroscopy Measurements

Introduction N ear infrared (NIR) spectroscopy is a powerful tool for measuring blood oxygenation and haemoglobin (Hb) content non-invasively. However, quantifi cation of these values in vivo is nearly impossible without an accurate measure of the optical pathlength. In highly-scattering media, such as biological tissue, photons take a tortuous and stochastic path through the sample and the effective pathlength can be many times the sample thickness or inter-optode spacing. Determination of the optical pathlength is complicated further by the fact that the angular distribution of scattered light intensity is wavelength-dependent. Thus, the mean pathlength is also wavelength-dependent. In order to determine haemoglobin content and oxygenation accurately then, it is crucial that the dependence of pathlength on wavelength be determined. This study introduced a novel method for measuring relative pathlength values in heart tissue using chromophores that exhibit multiple absorptions in the visible (vis)/ NIR spectrum. Since absorption intensity is proportional to pathlength, the relative absorption intensities for a single chromophore in vivo can be interpreted to yield relative pathlength values at the absorption wavelengths. Most previous studies of optical pathlength have used time-resolved methods and compared the values at only two or three wavelengths. With our method, multiple absorption peaks exhibited by a particular chromophore can yield greater pathlength data. Neodymium (Nd) is a lanthanide metal similar to those which have been used clinically as contrast agents in magnetic resonance imaging studies and, when bound to diethylene-triamine-pentaacetic acid (DTPA), it is non-toxic with only minimal alteration to its absorbance spectrum. It features six distinct absorption peaks between 500 nm and 900 nm (Figure 1), each with a full width at half maximum of less than 20 nm, permitting relative pathlength calculation data at those six wavelengths. Moreover, by using the water absorptions at 840 nm and 970 nm and the known water content of the tissue, this method yields absolute pathlength values at seven different wavelengths in the vis/NIR spectrum.