Simultaneous Measurement Of Changes Research Articles

Supplementary File: Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells

Supplementary File: Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells

Supplementary File: Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells

Supplementary File: Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells

Simultaneous Measurement of Changes in Mitochondrial and Endoplasmic Reticulum Free Calcium in Pancreatic Beta Cells.

The free calcium (Ca2+) levels in pancreatic beta cell organelles have been the subject of many recent investigations. Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The Kds of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 μM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.

In vitro circulation model driven by tissue-engineered dome-shaped cardiac tissue

The heart is an essential organ for animals and humans. With the increased availability of pluripotent stem cells, the use of three-dimensional cardiac tissues consisting of cultured cardiomyocytes in in vitro drug evaluation has been widely studied. Several models have been proposed for the realization of the pump function, which is the original function of the heart. However, there are no models that simulate the human circulatory system using cultured cardiac tissue. This study shows that a dome-shaped cardiac tissue fabricated using the cell sheet stacking technique can achieve a heart-like pump function and circulate culture medium, there by mimicking the human circulatory system. Firstly, human induced pluripotent stem cells were differentiated into autonomously beating cardiomyocytes, and cardiomyocyte cell sheets were created using temperature-responsive culture dishes. A cardiomyocyte sheet and a human dermal fibroblast sheet were stacked using a cell sheet manipulator. This two-layered cell sheet was then inflated to create a dome-shaped cardiac tissue with a base diameter of 8 mm. The volume of the dome-shaped cardiac tissue changed according to the autonomous beating. The stroke volume increased with the culture period and reached 21 ± 8.9 μl (n = 6) on day 21. It also responded to β-stimulant and extracellular calcium concentrations. Internal pressure fluctuations were also recorded under isovolumetric conditions by dedicated culture devices. The peak heights of pulsatile pressure were 0.33 ± 0.048 mmHg (n = 3) under a basal pressure of 0.5 mmHg on day 19. When the tissue was connected to a flow path that had check valves applied, it drove a directional flow with an average flow rate of approximately 1 μl s−1. Furthermore, pressure–volume (P–V) diagrams were created from the simultaneous measurement of changes in pressure and volume under three conditions of fluidic resistance. In conclusion, this cardiac model can potentially be used for biological pumps that drive multi-organ chips and for more accurate in vitro drug evaluation using P–V diagrams.

Real-time label-free assessment of T7 DNA polymerase immobilization

Immobilization of DNA-modifying enzymes on any surface is still a complex and challenging task in biotechnology and biosensorics. Therefore, this task very often is crucial, especially in biosensors dedicated to continuous monitoring of DNA. The novelty and the importance of this study are related to the development of continuously operating biosensors based on DNA-modifying enzymes. In this research we focused on the real-time monitoring of T7 DNA polymerase immobilization. Quartz crystal microbalance (QCM) was applied for the monitoring of immobilization of T7 DNA polymerase and assessment of analytical signals generated during the action of this enzyme. We have evaluated the covalent attachment of T7 DNA polymerase to different self-assembled monolayers consisting of different compositions of thiols, which were assembled on a gold-based surface of a QCM-resonator. To eliminate various aspects of inefficient T7 DNA polymerase immobilization and other inactivating factors associated with the inactivation of this DNA-enzyme, we have applied a strategy based on the protection of the exonuclease active site. We have examined the time-resolved simultaneous measurement of changes in frequency, energy dissipation, and surface saturation by T7 DNA polymerase during the immobilization process. Here reported findings suggest that detecting a low concentration of target DNA molecules can be achieved what enables efficient reuse of expensive DNA enzymes.

Blood pressure wave propagation—a multisensor setup for cerebral autoregulation studies

Objective. Cerebral autoregulation is critically important to maintain proper brain perfusion and supply the brain with oxygenated blood. Non-invasive measures of blood pressure (BP) are critical in assessing cerebral autoregulation. Wave propagation velocity may be a useful technique to estimate BP but the effect of the location of the sensors on the readings has not been thoroughly examined. In this paper, we were interested in studying whether the propagation velocity of a pressure wave in the direction from the heart to the brain may differ compared with propagation from the heart to the periphery, as well as across different physiological tasks and/or health conditions. Using non-invasive sensors simultaneously placed at different locations of the human body allows for the study of how the propagation velocity of the pressure wave, based on pulse transit time (PTT), varies across different directions. Approach. We present a multi-sensor BP wave propagation measurement setup intended for cerebral autoregulation studies. The presented sensor setup consists of three sensors, one placed on each of the neck, chest and finger, allowing simultaneous measurement of changes in BP propagation velocity towards the brain and to the periphery. We show how commonly tested physiological tasks affect the relative changes of PTT and correlations with BP. Main results. We observed that during maximal blow, valsalva and breath hold breathing tasks, the relative changes of PTT were higher when PTT was measured in the direction from the heart to the brain than from the heart to the peripherals. In contrast, during a deep breathing task, the relative change in PTT from the heart to the brain was lower. In addition, we present a short literature review of the PTT methods used in brain research. Significance. These preliminary data suggest that the physiological task and direction of PTT measurement may affect relative PTT changes. The presented three-sensor setup provides an easy and neuroimaging compatible method for cerebral autoregulation studies by allowing measurement of BP wave propagation velocity towards the brain versus towards the periphery.

Simultaneous Measurement of Changes in Neutrophil Granulocyte Membrane Potential, Intracellular pH, and Cell Size by Multiparametric Flow Cytometry

Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these important changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

Estimation of viscoelasticity of radial artery via simultaneous measurement of changes in pressure and diameter using a single ultrasound probe

To establish an evaluation index for vascular endothelial function, we developed an ultrasonic probe that can measure changes in blood pressure and blood vessel diameter at the same position in the radial artery. Based on phantom experiments, the pressure waveforms measured using the piezoelectric effect of the ultrasonic probe element and those using a pressure sensor exhibited a high correlation (correlation coefficient: r = 0.979 – 0.999). We confirmed the continuous measurement of the relationship between changes in blood pressure and the diameter from the in vivo experiments. We could then estimate the changes in viscoelasticity by calibrating the output from the probe element to the absolute blood pressure values in advance. The average coefficients of variations were 0.02 and 0.24 for elasticity and viscosity, respectively. This study demonstrated the possibility of measuring changes in the viscoelastic moduli of the radial arterial wall due to flow-mediated dilation using the developed ultrasonic probe.

Double-blind, randomised, placebo-controlled trial on the effect of L-carnitine and L-acetylcarnitine on sperm parameters in men with idiopathic oligoasthenozoospermia.

Carnitine is essential for energy metabolism and spermatozoa maturation. Combining L‐carnitine and L‐acetylcarnitine with micronutrients has been investigated as a treatment for infertility in men. We evaluated the effects of a therapeutic formulation, Proxeed Plus, on sperm parameters in oligoasthenozoospermic men. This prospective, randomised, double‐blind, placebo‐controlled clinical trial involved 175 males (19–44 years) with idiopathic oligoasthenozoospermia who failed to impregnate their partners (12 months). Males received Proxeed Plus or placebo for 3 and 6 months. Sperm volume, progressive motility and vitality significantly (p < 0.001) improved after 6 months compared to baseline. Sperm DNA fragmentation index significantly decreased compared to baseline (p < 0.001) and the 3‐month therapy (p = 0.014) in treated men. Increased seminal carnitine and α‐glucosidase concentration also positively correlated with improved progressive motility. Decreased DNA fragmentation index was the good predictor of progressive sperm motility >10%, and simultaneous measurement of changes in sperm vitality and DNA fragmentation index gave the highest probability of sperm motility 10% (AUC = 0.924; 95% CI = 0.852–0.996; p < 0.001). Logistic regression analyses revealed DNA fragmentation index decrease as the only independent predictor of sperm motility 10% (OR = 1.106; p = 0.034). We have demonstrated the beneficial effects of carnitine derivatives on progressive motility, vitality and sperm DNA fragmentation. Combining metabolic and micronutritive factors is beneficial for male infertility.

Open-field PET: Simultaneous brain functional imaging and behavioural response measurements in freely moving small animals

A comprehensive understanding of how the brain responds to a changing environment requires techniques capable of recording functional outputs at the whole-brain level in response to external stimuli. Positron emission tomography (PET) is an exquisitely sensitive technique for imaging brain function but the need for anaesthesia to avoid motion artefacts precludes concurrent behavioural response studies. Here, we report a technique that combines motion-compensated PET with a robotically-controlled animal enclosure to enable simultaneous brain imaging and behavioural recordings in unrestrained small animals. The technique was used to measure in vivo displacement of [11C]raclopride from dopamine D2 receptors (D2R) concurrently with changes in the behaviour of awake, freely moving rats following administration of unlabelled raclopride or amphetamine. The timing and magnitude of [11C]raclopride displacement from D2R were reliably estimated and, in the case of amphetamine, these changes coincided with a marked increase in stereotyped behaviours and hyper-locomotion. The technique, therefore, allows simultaneous measurement of changes in brain function and behavioural responses to external stimuli in conscious unrestrained animals, giving rise to important applications in behavioural neuroscience.

Linearity of electrical impedance tomography during maximum effort breathing and forced expiration maneuvers

Electrical impedance tomography (EIT) provides global and regional information about ventilation by means of relative changes in electrical impedance measured with electrodes placed around the thorax. In combination with lung function tests, e.g. spirometry and body plethysmography, regional information about lung ventilation can be achieved. Impedance changes strictly correlate with lung volume during tidal breathing and mechanical ventilation. Initial studies presumed a correlation also during forced expiration maneuvers. To quantify the validity of this correlation in extreme lung volume changes during forced breathing, a measurement system was set up and applied on seven lung-healthy volunteers. Simultaneous measurements of changes in lung volume using EIT imaging and pneumotachography were obtained with different breathing patterns. Data was divided into a synchronizing phase (spontaneous breathing) and a test phase (maximum effort breathing and forced maneuvers). The EIT impedance changes correlate strictly with spirometric data during slow breathing with increasing and maximum effort () and during forced expiration maneuvers (). Strong correlations in spirometric volume parameters (), /FVC (), and flow parameters PEF, , , () were observed. According to the linearity during forced expiration maneuvers, EIT can be used during pulmonary function testing in combination with spirometry for visualisation of regional lung ventilation.

Metal to phosphorus stoichiometries for freshwater phytoplankton in three remote lakes.

Simultaneous measurements of changes in phytoplankton biomass and the metal and phosphorus (P) content of cells have been captured to attest to metal to P stoichiometries for freshwater phytoplankton. Three Scottish lakes that had received high, medium or low metal contamination from the atmosphere were selected for study. Phytoplankton cells were collected and Inductively Coupled Plasma-Mass Spectrometry was used to measure their lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu), zinc (Zn), nickel (Ni), chromium (Cr), manganese (Mn), cobalt (Co) and P content. Increased phytoplankton growth in the lakes resulted in significant algae growth dilution of the mass-specific Pb, Cd, Hg, Cu, Ni and Cr in the phytoplankton. Changes in the phytoplankton cell count and their Hg, Pb, Cd, Cu, Mn, Co, Ni and Cr concentrations showed the process of algae bloom dilution to be subject to exponential decay, which accelerated in the order of Mn < Cu < Ni < Pb and Cd < Cr and Hg < Co. This indicated a metabolic and detoxification mechanism was involved in the active selection of metals. For the first time simultaneous measurements of metals and P stoichiometry in freshwater phytoplankton are reported. The mean metal to P stoichiometry generated was (C106P1N16)1000Pb0.019Hg0.00004Cu0.013Cd0.005Cr0.2Co0.0008Mn0.2Ni0.012 based on field measurements and the Redfield average C, N and P stoichiometry of (CH2O)106(NH3)16H3PO4.

Simultaneous High-Precision Imaging of Local Calcium and Single Sarcomere Length in Rat Neonatal Cardiomyocytes via Expression of Yellow Cameleon-Nano140 in Z-Discs

In the present study, we developed a novel experimental system for simultaneous measurement of changes in the intracellular Ca2+ concentration ([Ca2+]i) at local areas of the T-tubules / Z-discs and single sarcomere length (SL) via expression of FRET-based Ca2+ indicator Yellow Cameleon (YC)-Nano140 in Z-discs (α-actinin) in rat neonatal cardiomyocytes. Under dual-view fluorescence microscopy, local [Ca2+]i was determined by the YFP/CFP fluorescence ratio and SL by the peak-to-peak distance of the YFP fluorescence profile (see Shintani et al., J Gen Physiol 2014 for SL nanometry: S.D., 12 nm in the present study). During spontaneous beating, although [Ca2+]i changed uniformly in a localized area (5 consecutive sarcomeres) of a myocyte, sarcomere shortening / lengthening was not synchronized, but rather occurred in an asynchronous fashion, suggesting that the length of each sarcomere is determined via tug-of-war forces of sarcomeres along the myofibril. Likewise, we successfully quantified amplifications of systolic [Ca2+]i and single sarcomere contraction upon β-adrenergic stimulation. Furthermore, α-actinin-YC-Nano140 enabled high-precision simultaneous measurements of local [Ca2+]i and single sarcomere contraction under electric field stimulation at 5 Hz. The present experimental system has a broad range of application possibilities for analyzing cardiac excitation-contraction (E-C) coupling in detail at the single sarcomere level. Here, we discuss 1) the technical aspects of the application of YC-Nano140 for the analysis of cardiac E-C coupling, and 2) mechanistic implications of single sarcomere dynamics in relation to local [Ca2+]i changes.

Multiple Reaction Monitoring Mode Based Liquid Chromatography-Mass Spectrometry Method for Simultaneous Quantification of Brassinolide and Other Plant Hormones Involved in Abiotic Stresses.

Plant hormones are the key regulators of adaptive stress response. Abiotic stresses such as drought and salt are known to affect the growth and productivity of plants. It is well known that the levels of plant hormones such as zeatin (ZA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and brassinolide (BR) fluctuate upon abiotic stress exposure. At present, there is not any single suitable liquid chromatography-mass spectrometry (LC-MS) method for simultaneous analysis of BR and other plant hormones involved in abiotic stresses. In the present study, we developed a simple, sensitive, and rapid method for simultaneous analysis of five major plant hormones, ZA, ABA, JA, SA, and BR, which are directly or indirectly involved in drought and salt stresses. The optimized extraction procedure was simple and easy to use for simultaneous measurement of these plant hormones in Arabidopsis thaliana. The developed method is highly reproducible and can be adapted for simultaneous measurement of changes in plant hormones (ZA, ABA, JA, SA, and BR) in response to abiotic stresses in plants like A. thaliana and tomato.

An experimental investigation on the drying kinetics of a single coarse particle of Belchatow lignite in an atmospheric superheated steam condition

The efficiency of lignite-fired power plants is sacrificed by the high moisture content of this energy carrier. The most adequate effort for upgrading the potential of lignite is the drying process, influenced by both the drying conditions and the physicochemical features of lignite. This paper presents studies on the drying kinetic of the Belchatow lignite originating from the biggest Polish lignite mine. Experimental attempts were conducted for spherical lignite samples dried in a superheated steam atmosphere at the temperature range of 110–170°C. Each experiment includes the simultaneous measurements of changes in weight and temperature profiles for a single sample. Additionally, the drying process was recorded to observe the cracking on the surface of the sample and its shrinkage. The kinetics were described in the form of moisture content, drying rate and temperature profiles over the drying process. The time and rate of the superheated steam drying process depending on the sample size and steam temperature were estimated. Those parameters are essential for the design of an effective industrial coal drying system, which allows for the latent heat recovery of water evaporation from the lignite, which in turn will improve the thermal efficiency of the lignite-fired power generation.

(18)F-MCL-524, an (18)F-Labeled Dopamine D2 and D3 Receptor Agonist Sensitive to Dopamine: A Preliminary PET Study.

PET has been used to examine changes in neurotransmitter concentrations in the living brain. Pioneering PET studies on the dopamine system have used D2 and D3 receptor (D2/D3) antagonists such as (11)C-raclopride. However, more recently developed agonist radioligands have shown enhanced sensitivity to endogenous dopamine. A limitation of available agonist radioligands is that they incorporate the short-lived radioisotope (11)C. In the current study, we developed the (18)F-labeled D2/D3 receptor agonist (R)-(-)-2-(18)F-fluoroethoxy-N-n-propylnorapomorphine ((18)F-MCL-524). In total, 10 PET measurements were conducted on 5 cynomolgus monkeys. Initially, the binding of (18)F-MCL-524 was compared with that of (11)C-MNPA in 3 monkeys. Second, the specificity of (18)F-MCL-524 binding was examined in pretreatment studies using raclopride (1.0 mg/kg) and d-amphetamine (1.0 mg/kg). Third, a preliminary kinetic analysis was performed using the radiometabolite-corrected arterial input function of the baseline studies. Finally, 2 whole-body PET measurements were conducted to evaluate biodistribution and radiation dosimetry after intravenous injection of (18)F-MCL-524. (18)F-MCL-524 entered the brain and provided striatum-to-cerebellum ratios suitable for reliable quantification of receptor binding using the multilinear reference tissue model. Mean striatal nondisplaceable binding potential (BPND) values were 2.0 after injection of (18)F-MCL-524 and 1.4 after (11)C-MNPA. The ratio of the BPND values of (18)F-MCL-524 and (11)C-MNPA was 1.5 across striatal subregions. After administration of raclopride and d-amphetamine, the (18)F-MCL-524 BPND values were reduced by 89% and 56%, respectively. Preliminary kinetic analysis demonstrated that BPND values obtained with the 1-tissue- and 2-tissue-compartment models were similar to values obtained with the multilinear reference tissue model. Estimated radiation doses were highest for gallbladder (0.27 mSv/MBq), upper large intestine (0.19 mSv/MBq), and small intestine (0.17 mSv/MBq). The estimated effective dose was 0.035 mSv/MBq. The (18)F-labeled agonist (18)F-MCL-524 appears suitable for quantification of D2/D3 receptor binding in vivo, and the results encourage extension to human studies. The longer half-life of (18)F makes (18)F-MCL-524 attractive for studies on modulation of the dopamine concentration-for example, in combination with simultaneous measurement of changes in blood-oxygen-level-dependent signal using bimodal PET/functional MRI.

Quartz Resonator for Simultaneous Measurement of Changes in Mass and Electrical Impedance during Protein Adsorption

A novel quartz resonator has been developed for the synchronized measurement of changes in mass and electrical impedance during the adsorption of various concentrations of bovine serum albumin (BSA). The top platinum surface of the quartz crystal was used as the working electrode and real-time Fourier transform analysis was conducted to obtain time-resolved electrochemical impedance spectra. The change in mass due to the BSA adsorption was calculated from the change in the frequency of the quartz resonator. These simultaneous measurements of mass and electrical impedance were used to determine not only the kinetics of protein adsorption but also the morphological changes in the adsorbed layer, which were confirmed with atomic force microscopy.

NAPPA based nanogravimetric biosensor: Preliminary characterization

We present our preliminary results about the implementation of a new nanogravimetric biosensor realized combining Quartz Crystal Microbalance with Dissipation Monitoring Nanogravimetry and an innovative protein cell-free expression system named Nucleic Acid Programmable Protein Arrays (NAPPA) that allowed us to immobilize on the quartz surface, as sensing molecule, the proteins starting from its plasmid. We herby present results about the characterization of the nanogravimetric biosensor, its sensitivity, and its selectivity. Moreover we demonstrated how the simultaneous measurements of changes in resonance frequency and in energy dissipation were fundamental to correctly interpret the behavior of complex system such as NAPPA.

An association between vasomotion and oxygen extraction.

Vasomotion is defined as a spontaneous local oscillation in vascular tone whose function is unclear but may have a beneficial effect on tissue oxygenation. Optical reflectance spectroscopy and laser Doppler fluximetry provide unique insights into the possible mechanisms of vasomotion in the cutaneous microcirculation through the simultaneous measurement of changes in concentration of oxyhemoglobin ([HbO(2)]), deoxyhemoglobin ([Hb]), and mean blood saturation (S(mb)O(2)) along with blood volume and flux. The effect of vasomotion at frequencies <0.02 Hz attributed to endothelial activity was studied in the dorsal forearm skin of 24 healthy males. Fourier analysis identified periodic fluctuations in S(mb)O(2) in 19 out of 24 subjects, predominantly where skin temperatures were >29.3°C (X(2) = 6.19, P < 0.02). A consistent minimum threshold in S(mb)O(2) (mean: 39.4%, range: 24.0-50.6%) was seen to precede a sudden transient surge in flux, inducing a fast rise in S(mb)O(2). The integral increase in flux correlated with the integral increase in [HbO(2)] (Pearson's correlation r(2) = 0.50, P < 0.001) and with little change in blood volume suggests vasodilation upstream, responding to a low S(mb)O(2) downstream. This transient surge in flux was followed by a sustained period where blood volume and flux remained relatively constant and a steady decrease in [HbO(2)] and equal and opposite increase in [Hb] was considered to provide a measure of oxygen extraction. A measure of this oxygen extraction has been approximated by the mean half-life of the decay in S(mb)O(2) during this period. A comparison of the mean half-life in the 8 normal subjects [body mass index (BMI) <26.0 kg/m(2)] of 12.2 s and the 11 obese subjects (BMI >29.5 kg/m(2)) of 18.8 s was statistically significant (Mann Whitney, P < 0.004). The S(mb)O(2) fluctuated spontaneously in this saw tooth manner by an average of 9.0% (range 4.0-16.2%) from mean S(mb)O(2) values ranging from 30 to 52%. These observations support the hypothesis that red blood cells may act as sensors of local tissue hypoxia, through the oxygenation status of the hemoglobin, and initiate improved local perfusion to the tissue through hypoxic vasodilation.

Simultaneous measurement of changes in light absorption due to the reduction of cytochrome c oxidase and light scattering in rat brains during loss of tissue viability

We performed the simultaneous measurement of intrinsic optical signals (IOSs) related to metabolic activity and cellular and subcellular morphological characteristics, i.e., light scattering for a rat global ischemic brain model made by rapidly removing blood by saline infusion. The signals were measured on the basis of multiwavelength diffuse reflectances in which 605 and 830 nm were used to detect the IOSs that are thought to be dominantly affected by redox changes of heme aa(3) and CuA in cytochrome c oxidase (CcO), respectively. For measuring the scattering signal, the wavelength that was found to be most insensitive to the absorption changes, e.g., approximately 620 nm, was used. The measurements suggested that an increase in the absorption due to reduction of heme aa(3) occurred soon after blood clearance, and this was followed by a large triphasic change in light scattering, during which time a decrease in the absorption due to reduction of CuA occurred. Through the triphasic scattering change, scattering signals increased by 5.2 +/- 1.5% (n = 5), and the increase in light scattering showed significant correlation with both the reflectance intensity changes at 605 and 830 nm. This suggests that morphological changes in cells correlate with reductions of heme aa(3) and CuA. Histological analysis of tissue after the triphasic scattering change showed no alteration in either the nuclei or the cytoskeleton, but electron microscopic observation revealed deformed, enlarged mitochondria and expanded dendrites. These findings suggest that the simultaneous measurement of absorption signals related to the redox changes in the CcO and the scattering signal is useful for monitoring tissue viability in the brain.