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PubMed2004

Experimental measurements for studying angular and spectral variation of thermal infrared emissivity.

One condition for precise multiangle algorithms for estimating sea and land surface temperature with the data from the Advanced Along Track Scanning Radiometer is accurate knowledge of the angular variation of surface emissivity in the thermal IR spectrum region. Today there are very few measurements of this variation. The present study is conducted to provide angular emissivity measurements for five representative samples (water, clay, sand, loam, gravel). The measurements are made in one thermal IR broadband (8-13 microm) and three narrower bands (8.2-9.2, 10.3-11.3, and 11.5-12.5 microm) at angles of 0 degrees-60 degrees (at 5 degrees increments) to the surface normal. The results show a general decrease in emissivity with increasing viewing angles, with the 8.2-9.2-microm channel the most sensitive to this dependence and sand the sample showing the greatest variation.

Journal Article↗
PubMed2001

Baroreflex and oscillation of heart period at 0.1 Hz studied by alpha-blockade and cross-spectral analysis in healthy humans.

1. Parameters derived from frequency-domain analysis of heart period and blood pressure variability are gaining increasing importance in clinical practice. However, the underlying physiological mechanisms in human subjects are not fully understood. Here we address the question as to whether the low frequency variability (approximately 0.1 Hz) of the heart period may depend on a baroreflex-mediated response to blood pressure oscillations, induced by the alpha-sympathetic drive on the peripheral resistance. 2. Heart period (ECG), finger arterial pressure (Finapres) and respiratory airflow were recorded in eight healthy volunteers in the supine position with metronome respiration at 0.25 Hz. We inhibited the vascular response to the sympathetic vasomotor activity with a peripheral alpha-blocker (urapidil) and maintained mean blood pressure at control levels with angiotensin II. 3. We performed spectral and cross-spectral analysis of heart period (RR) and systolic pressure to quantify the power of low- and high-frequency oscillations, phase shift, coherence and transfer function gain. 4. In control conditions, spectral analysis yielded typical results. In the low-frequency range, cross-spectral analysis showed high coherence (> 0.5) and a negative phase shift (-65.1 +/- 18 deg) between RR and systolic pressure, which indicates a 1-2 s lag in heart period changes in relation to pressure. In the high-frequency region, the phase shift was close to zero, indicating simultaneous fluctuations of RR and systolic pressure. During urapidil + angiotensin II infusion the low-frequency oscillations of both blood pressure and heart period were abolished in five cases. In the remaining three cases they were substantially reduced and lost their typical cross-spectral characteristics. 5. We conclude that in supine rest conditions, the oscillation of RR at low frequency is almost entirely accounted for by a baroreflex mechanism, since it is not produced in the absence of a 0.1 Hz pressure oscillation. 6. The results provide physiological support for the use of non-invasive estimates of the closed-loop baroreflex gain from cross-spectral analysis of blood pressure and heart period variability in the 0.1 Hz range.

Adrenergic alpha-Antagonists↗
PubMed1998

Assessing baroreflex sensitivity in post-myocardial infarction patients: comparison of spectral and phenylephrine techniques.

OBJECTIVES: This study sought to compare, in post-myocardial infarction patients, baroreflex sensitivity (BRS) measured by the phenylephrine method (Phe-BRS) with that estimated by the Robbe (Robbe-BRS) and Pagani (alpha-low frequency [LF] and alpha-high frequency [HF]) spectral techniques. BACKGROUND: BRS assessed by Phe-BRS has been shown to be of prognostic value in patients with a previous myocardial infarction, but the need for drug injection limits the use of this technique. Several noninvasive methods based on spectral analysis of systolic arterial pressure and heart period have been proposed, but their agreement with Phe-BRS has never been investigated in post-myocardial infarction patients. METHODS: The linear association and the agreement between each spectral measurement and Phe-BRS were assessed by correlation analysis and by computing the relative bias and the limits of agreement in 51 post-myocardial infarction patients. RESULTS: The correlation with Phe-BRS was r = 0.63 for Robbe-BRS, r = 0.62 for alpha-LF and r = 0.59 for alpha-HF. The relative bias was significant for alpha-LF (2.6 ms/mm Hg, p < 0.001) and alpha-HF (2.5 ms/mm Hg, p = 0.01) and not significant (-0.6 ms/mm Hg, p = 0.3) for Robbe-BRS. The normalized limits of agreement ranged from -98% to 95% for Robbe-BRS, from -67% to 126% for alpha-LF and from -108% to 143% for alpha-HF. When patients were classified according to left ventricular ejection fraction (LVEF, cutoff value 40%), the relative bias was higher in patients with a depressed LVEF, although statistical significance was high only for Robbe-BRS and was borderline for alpha-LF. The limits of agreement were similar in both groups of patients (p > 0.3). CONCLUSIONS: Despite a substantial linear association, the agreement between spectral measurements and Phe-BRS in post-myocardial infarction patients is weak because the difference can be as large as the BRS value being estimated. Phe-BRS is the measurement most associated with hemodynamic impairment. Because several factors within each method contribute to the overall difference, neither method can be defined as being better than the other in estimating baroreflex gain, nor can one be used as an alternative to the other. Ad hoc studies are needed to assess which method provides the most useful physiologic or pathophysiologic information or the most accurate prediction of prognosis.

Baroreflex↗
PubMed2004

Bayesian estimation for species identification in single-molecule fluorescence microscopy.

In this article we describe a recursive Bayesian estimator for the identification of diffusing fluorophores using photon arrival-time data from a single spectral channel. We present derivations for all relevant diffusion and fluorescence models, and we use simulated diffusion trajectories and photon streams to evaluate the estimator's performance. We consider simplified estimation schemes that bin the photon counts within time intervals of fixed duration, and show that they can perform well in realistic parameter regimes. The latter results indicate the feasibility of performing identification experiments in real time. It will be straightforward to generalize our approach for use in more complicated scenarios, e.g., with multiple spectral channels or fast photophysical dynamics.

Algorithms↗
PubMed1999

Modeling of polychromatic attenuation using computed tomography reconstructed images.

This paper presents a procedure for estimating an accurate model of the CT imaging process including spectral effects. As raw projection data are typically unavailable to the end-user, we adopt a post-processing approach that utilizes the reconstructed images themselves. This approach includes errors from x-ray scatter and the nonidealities of the built-in soft tissue correction into the beam characteristics, which is crucial to beam hardening correction algorithms that are designed to be applied directly to CT reconstructed images. We formulate this approach as a quadratic programming problem and propose two different methods, dimension reduction and regularization, to overcome ill conditioning in the model. For the regularization method we use a statistical procedure, Cross Validation, to select the regularization parameter. We have constructed step-wedge phantoms to estimate the effective beam spectrum of a GE CT-I scanner. Using the derived spectrum, we computed the attenuation ratios for the wedge phantoms and found that the worst case modeling error is less than 3% of the corresponding attenuation ratio. We have also built two test (hybrid) phantoms to evaluate the effective spectrum. Based on these test phantoms, we have shown that the effective beam spectrum provides an accurate model for the CT imaging process. Last, we used a simple beam hardening correction experiment to demonstrate the effectiveness of the estimated beam profile for removing beam hardening artifacts. We hope that this estimation procedure will encourage more independent research on beam hardening corrections and will lead to the development of application-specific beam hardening correction algorithms.

Algorithms↗
PubMed1995

A data correction method for surface measurement of vibration on the human body.

A data correction method to eliminate the effect of local tissue-accelerometer vibration from surface measurements of vibration over the spine has been developed and compared with previous direct measurements. A single degree-of-freedom linear model for the local tissue-accelerometer system in the vertical and the fore-and-aft axes is assumed. The natural frequency and the damping ratio of the local system are estimated so as to form a correction frequency function, using spectral analysis of the free vibration response of the local system caused by transient displacements of the accelerometer attached to the body surface. Accelerometers were attached to the skin over the spinous process of the vertebra L3 and on the abdominal wall. For four different masses and each site, correction frequency functions were computed. Seated subjects were then exposed to vertical random vibration (0.5-35 Hz) and acceleration transfer functions from the seat to each accelerometer were calculated. Different transfer functions were obtained with different additional masses but the differences were eliminated by the correction method so as to indicate the transfer functions to the spine and the viscera. For vertical responses, the correction method was effective at frequencies below the estimated natural frequencies of the local system. Fore-and-aft response over the spine did not require correction at frequencies below 35 Hz.

Abdominal Muscles↗
PubMed2000

Early detection of cardiovascular autonomic neuropathy in diabetic pigs using blood pressure and heart rate variability.

Cardiac autonomic neuropathy is a common complication in insulin dependent diabetes mellitus. Nevertheless, little is known about when this impairment occurs during the time course of the disease. Analysis of blood pressure (BP) and heart rate (HR) variability could be used to detect early signs of autonomic alteration. To test this proposal, twelve sexually mature male Yucatan miniature pigs were equipped with an arterial catheter for telemetric BP analysis, and with a venous access. BP and HR were recorded together with respiratory movements while the animals were resting in a sling. After the first recording session performed when the pigs were 5 months old, streptozotocin (STZ) was used to induce diabetes in seven pigs, while the five others were controls. BP and HR were measured 3 and 6 months after the onset of diabetes and at a similar age in the controls. BP and HR oscillated at the respiratory range (0.19 Hz). Spectral analysis showed this respiratory component was the main determinant of the short-term variability of BP and HR. Atropine increased HR and BP and markedly diminished the respiratory sinus arrhythmia. Propranolol diminished HR and the respiratory peak of HR. A reduced respiratory oscillation of BP paralleled the diminution of the respiratory peak of HR. Baroreceptor-HR reflex was estimated using injections of phenylephrine and nitroprusside, and by cross-spectral analysis between BP and HR. Atropine shifted the curve to higher HR values, while propranolol reduced the level of the upper plateau. Atropine decreased both the coherence and gain of the cross-spectral analysis. STZ injection resulted in a type 1 diabetes. At 3 months, diabetic pigs exhibited low levels of BP and a reduced overall variability of HR and BP. Spectral analysis indicated the respiratory sinus arrhythmia was markedly reduced. In addition, the sensitivity of the baroreceptor-HR reflex was reduced. At a latter stage of diabetes these alterations were marked and the level of the resting HR was increased. These data demonstrate the dual (vagal and sympathetic) control of HR in pigs and the dominant role of respiration in the genesis of HR and BP fluctuations. The spectral and cross-spectral analysis of BP and HR were altered after 3 months of diabetes and could be proposed as early detectors of cardiac autonomic neuropathy.

Adrenergic beta-Antagonists↗
PubMed1976

Noninvasive ultrasonic carotid angiography: prospective validation by contrast arteriography.

Pulsed ultrasonic images of the carotid bifurcation in 82 vessels of 43 patients were compared independently with contrast arteriograms for stenosis (by percentage quartiles) or occlusion of the internal carotid artery. All 14 occluded vessels were identified correctly by ultrasound but were visualized on repeat examination. Estimation of percentage stenosis on ultrasonic images agreed with the quartile determination by contrast arteriography in 35 of 68 (51 percent) vessels and was within one quartile of correct interpretation in 48 of 68 (71 percent). The interpretative error of grading stenosis of ultrasonic images was due to vascular wall calcification which inhibited ultrasound transmission. This limitation was overcome by sound spectral (sonographic) analysis of distal internal carotid flow velocity which allowed estimation of stenosis within one quartile of that determined by contrast arteriography in 46 of 47 (98 percent) vessels.

Adult↗
PubMed2000

Stability of the noninvasive baroreflex sensitivity assessment using cross-spectral analysis of heart rate and arterial blood pressure variabilities.

BACKGROUND: Depressed baroreflex sensitivity (BRS), usually estimated using the invasive phenylephrine method or the nitroprusside test, is significantly and independently associated with an increased risk of malignant ventricular arrhythmias and sudden cardiac death in patients surviving acute myocardial infarction. Several investigators have compared the standard phenylephrine test and different noninvasive methods. HYPOTHESIS: This study evaluated the influence of different body positions with different breathing regimes on cross-spectral baroreflex indices (coherence between the spectral densities of blood pressure and cardiac cycle variabilities) in both low- and high-frequency bands. METHODS: The data were obtained in 103 patients (73 males, aged 53 +/- 12 years) with coronary artery disease and/or hypertension. Simultaneous electrocardiographic and noninvasive blood pressure recordings were obtained in each subject in both supine and sitting positions during both spontaneous and slow and fast controlled respiration (0.1 and 0.33 Hz). RESULTS: The results show a significant bias and disagreement between noninvasive baroreflex sensitivity (BRS) indices. The mean values of the baroreflex in low frequency ranged from 5.0 +/- 5.3 to 10.1 +/- 7.9 ms/mmHg, while in high frequency, the mean values ranged from 6.6 +/- 6.1 to 10.1 +/- 7.9 ms/mmHg. The limits of agreement ranged from +/-1.7 to +/-4.1 ms/mmHg with bias from -1.0 to +0.7 ms/mmHg. CONCLUSION: A comprehensive comparison of different methods shows that BRS estimated in low-frequency band in sitting position during spontaneous respiration is the most representative part of the global baroreflex gain.

Adult↗
PubMed1999

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells.

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1-2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.

Cell Nucleus↗
PubMed2004

[Estimation of autonomic nervous system tension in patients with syncope of unknown origin--traps in clinical interpretation results of heart rate variability].

UNLABELLED: Voluntary character of a choice of statistical methods weakens the reliability of the results of heart rate variability assessment and based on them clinical conclusions. The aim of the study was to estimate reliability of the chosen statistical methods of analysing changes in spectral components during tilt testing (TT). MATERIAL AND METHODS: 62 patients (34 women), the average age 38.8 +/- 12.8 years, with at least 2 syncopes during the last 6 months that had not been diagnosed by other examinations, were enrolled. The control group consisted of 44 age- and sex-matched volunteers (21 women), the average age 37.4 +/- 12.6 years, who had no history of syncope. All subjects underwent tilt testing according to Westminster protocol. If the result of a passive phase was negative, 0.25 mg nitroglycerine was applied sublingually and the examination was continued for the next 20 minutes. The type of syncopal reaction was defined according to VASIS classification (the vasovagal syncope international study). The evaluation of heart rate variability was based on the frequency domain methods. The low frequency (LF), the high frequency (HF) component and the balance (the LF/HF ratio) were estimated. The response of the autonomic nervous system (AUN) to tilting was evaluated by tests comparing mean values of respective components (the quantitative analysis) and by the modulation index (deltaXFm) reflecting, in percentage terms, changes in analyzed variable values. Taking into account the obtained results there was also a qualitative analysis performed. This analysis referred to the modulation index, which reflected increase or decrease in spectral component value. RESULTS: Tilt testing explained vasovagal origin of syncope in 35 (60%) patients with syncope. Patients were divided into 3 groups according to anamnesis data and results of tilt testing. Group I consisted of 42 healthy volunteers who had negative result of TT Group II--35 patients with history of syncopes and positive result of TT, and Group III--23 patients with history of syncopes and negative result of TT. In all study groups the quantitative analysis revealed the decrease in the LF and HF component values, whereas the evaluation of autonomic nervous system reactivity based on deltaXFm showed the increase in LF power, concomitant with strongly marked, nearly uniform decrease in HF power in Group I and II. The results obtained from the qualitative analysis did not reveal statistically significant differences between study groups in distribution of defined types of autonomic nervous system reactions. CONCLUSIONS: 1. The quantitative analysis has limited credibility in clinical interpretation of changes in autonomic nervous system activity (especially in the low frequency component--LF) occurring during tilt testing. 2. The best estimation of autonomic nervous system activity during tilt testing is assured by the modulation index supplemented by the preliminary selection of data, i.e., rejection of extreme values or those rarely occurring qualitative changes in spectral components.

Adolescent↗
PubMedDate not supplied

Determination of regurgitant flow and volume by integrating actual proximal velocities over hemispheres (IPROV) in two orthogonal planes.

The proximal acceleration technique is a promising technique for quantification of regurgitant valve flow. Although the shape of the regurgitant proximal isovelocity field has been shown to vary with orifice size, geometry, and driving pressure, normally the centerline velocity alone is used for estimation of flow. In this model study of pulsatile flow, two-dimensional and spectral Doppler data were transferred digitally to a computer in which proximal velocity fields were corrected for time and angle errors. With the purpose of improving accuracy, flow was estimated by integrating proximal velocities over nonisovelocity spheric control surfaces in the best zone of measurement (0.15 to 0.45 m/sec at an angle up to +/- 45 degrees from the center line) in two perpendicular planes. Three regurgitant volumes in the range of 5 to 21 ml were studied for circular (diameters of 4, 6, and 8 mm), crescent, and diagonal orifices. The quotient between effective orifice area, estimated by dividing peak flow with peak velocity in the vena contracta, and true orifice area (Aeff = Q(tm)/Vo(tm)) was 0.66 (range 0.60 to 0.79), 0.50 (0.48 to 0.52), and 0.67 (0.66 to 0.68) for the circular, crescent, and diagonal orifices, respectively. Regurgitant volume estimated by multiplying effective orifice area by the velocity-time integral in the vena contracta (V = Aeff.velocity-time integral) ranged from 92% to 115% of the true volume for the circular, 89% to 92% for the crescent, and 105% to 112% for the diagonal orifices, respectively. It is possible to calculate regurgitant volume correctly with data acquisition from multiple hemispheres and planes and postprocessing of data. This amendment of the proximal acceleration technique has great advantage over the center-line method, especially when the orifice is asymmetric.

Blood Flow Velocity↗
PubMed2003

Chlorophyll content in eucalypt vegetation at the leaf and canopy scales as derived from high resolution spectral data.

The physiological status of forest canopy foliage is influenced by a range of factors that affect leaf pigment content and function. Recently, several indices have been developed from remotely sensed data that attempt to provide robust estimates of leaf chlorophyll content. These indices have been developed from either hand-held spectroradiometer spectra or high spectral resolution (or hyperspectral) imagery. We determined if two previously published indices (Datt 1999), which were specifically developed to predict chlorophyll content in eucalypt vegetation by remote sensing at the leaf scale, can be extrapolated accurately to the canopy. We derived the two indices from hand-held spectroradiometer data of eucalypt leaves exhibiting a range of insect damage symptoms. We also derived the indices from spectra obtained from high spectral and spatial resolution Compact Airborne Spectrographic Imager 2 (CASI-2) imagery to determine if reasonable estimates at a scale of < 1 m can be achieved. One of the indices (R 850/R 710 index, where R is reflectance) derived from hand-held spectroradiometer data showed a moderate correlation with relative leaf chlorophyll content (r = 0.59, P < 0.05) for all dominant eucalypt species in the study area. The R (850)/R (710) index derived from CASI-2 imagery yielded slightly lower correlations over the entire data set (r = 0.42, P < 0.05), but correlations for individual species were high (r = 0.77, P < 0.05). A scaling analysis indicated that the R (850)/R (710) index was strongly affected by soil and water cover types when pixels were mixed, but appeared to be invariant to changes in proportions of understory, which may limit its application.

Chlorophyll↗
PubMed1980

pH-induced alterations in the structure and activity of cytochrome c oxidase.

The effects of pH on the activity and structure of beef heart cytochrome c oxidase have been studied in the pH range 5.0-7.6. (i) A group with pK of approximately 5.45 has been readily detected in the pH vs. activity curve. This group must be deprotonated to achieve maximal activity. (ii) A group with a similar pK (5.45) has been detected and contributes to the spectral character of the reduced oxidase. Over the range pH 5.0-7.6 no other acid-sensitive group contributes to the spectrum of the reduced oxidase. (iii) The oxidized oxidase shows at least three acid-sensitive groups contributing to the spectrum. One occurs in the pH 7 range and another in the pH 5.6 range; below pH 5.2 additional pH-sensitive groups are apparent. Accurate estimation of the pK's of the groups responsible for the spectral changes in the oxidized oxidase has not been possible. (iv) The spectrum of the "oxygenated" (428 nm) conformer of the oxidized protein is invariant over the range ph 5.5-7. (v) The changes occurring in the spectrum of the purified oxidase also occur in the protein contained in phospholipid vesicles. (vi) The data are discussed in terms of the mechanism by which the oxidase, during its in situ catalytic cycle, may give rise to the primary events in energy coupling.

Animals↗
PubMedDate not supplied

Robust spectrotemporal reverse correlation for the auditory system: optimizing stimulus design.

The spectrotemporal receptive field (STRF) is a functional descriptor of the linear processing of time-varying acoustic spectra by the auditory system. By cross-correlating sustained neuronal activity with the dynamic spectrum of a spectrotemporally rich stimulus ensemble, one obtains an estimate of the STRF. In this article, the relationship between the spectrotemporal structure of any given stimulus and the quality of the STRF estimate is explored and exploited. Invoking the Fourier theorem, arbitrary dynamic spectra are described as sums of basic sinusoidal components--that is, moving ripples. Accurate estimation is found to be especially reliant on the prominence of components whose spectral and temporal characteristics are of relevance to the auditory locus under study and is sensitive to the phase relationships between components with identical temporal signatures. These and other observations have guided the development and use of stimuli with deterministic dynamic spectra composed of the superposition of many temporally orthogonal moving ripples having a restricted, relevant range of spectral scales and temporal rates. The method, termed sum-of-ripples, is similar in spirit to the white-noise approach but enjoys the same practical advantages--which equate to faster and more accurate estimation--attributable to the time-domain sum-of-sinusoids method previously employed in vision research. Application of the method is exemplified with both modeled data and experimental data from ferret primary auditory cortex (AI).

Acoustic Stimulation↗
PubMed1982

Spectral bandwidths of color-opponent cells of geniculocortical pathway of macaque monkeys.

1. The spectral-response bandwidth and peak sensitivity of the responses of color-opponent retinal ganglion cells of the macaque monkey were examined in conditions of neutral adaptation. Color-opponent cells showed specific "signatures" in plots of response bandwidth versus wavelength of the peak sensitivity that allow for an acceptable estimate of the cone types whose signals mediate cell responses. 2. Averaged spectral bandwidths of color-opponent ganglion cells were compared with published data from color-selective neurons of subsequent levels of the geniculocortical pathway, including the extrastriate area termed V4. No significant differences were found between color-selective cells of the retina, dorsal lateral geniculate body, striate cortex, and V4. 3. The spectral location of the peak sensitivity of responses of the various types of color-opponent ganglion cells showed a relatively broad distribution, loosely clustering at some spectral loci. Comparison of such distribution with that of recently reported V4 cells response indicates that a remarkable scarcity of "blue/yellow" opponent responses in such reports. 4. In association with more recent electrophysiological studies of V4 cells, the results do not support current claims of a color specialization of this extrastriate area, and suggest lack of significant spectral tuning of the retinal output in, so far known, higher visual centers having color-selective cells.

Animals↗
PubMed1990

Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity.

The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population. We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492-8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.

Binding Sites↗