Age-related hair denaturation related to protein carbonyls.

OBJECTIVE: Hair ageing is accompanied by hair fibres becoming irregularly shaped causing them to stick out in irregular directions or have more curliness and being spread out. This is believed to be due to changes within the hair fibre structure which occur with ageing, and one of the causes of these changes could be an increase in the number of protein carbonyl groups present in the hair. The aim of this study is to investigate the internal denaturation of hair related to protein carbonyls in attempt to gain new insight into age-related changes that occur in hair. METHODS: The degree of carbonylation of the hair structural protein as determined by fluorescent labelling and Western blotting analysis was used to investigate the primary structure of hair protein. The amount of helix, a common conformation in the secondary structure of proteins, in hair in groups of women with different ages was also analysed using infrared microscopy coupled with multivariate curve resolution (MCR). From the results of this, an image of the two-dimensional distribution of the α-helices was generated for the hair taken from each age group. Also, high-pressure differential scanning calorimetry (HPDSC) of the hair in water was performed on the hair taken from each age group to determine the peak temperature of endothermic effect and the enthalpy of denaturation. RESULTS: We found that the amino group content in hair proteins decreased and Type II keratin, one of the subunits of intermediate filament, was more carbonylated with age. The results of the MCR indicated eight separate components, including components of the secondary structure of proteins, such as α helices and ß sheets. Two-dimensional images of the hair cross-sections revealed that the presence of α helices decreased with age. In addition, data from the HPDSC showed that the enthalpy associated with the denaturing temperature also significantly decreased with age. CONCLUSION: These results suggest that there is a negative correlation between age and structural integrity of the helix segment in intermediate filament. The results of this study also show that there is a positive correlation between age-related hair denaturation and protein carbonyls.

OBJECTIF: Le vieillissement du cheveu s'accompagne d'une irrégularité de la forme des fibres capillaires, ce qui les fait ressortir dans des directions irrégulières ou les rend plus bouclées, et d'un étalement de celles-ci. On pense que cela est dû à des changements dans la structure des fibres capillaires qui surviennent avec l'âge, et l'une des causes de ces changements pourrait être une augmentation du nombre de protéines carbonyles présentes dans le cheveu. L'objectif de cette étude est d'étudier la dénaturation interne du cheveu liée aux protéines carbonyles afin d'obtenir de nouvelles informations sur les changements liés à l'âge qui surviennent dans le cheveu. MÉTHODES: Le degré de carbonylation de la protéine structurelle du cheveu, déterminé par marquage par fluorescence et analyse par western blotting, a été utilisé pour étudier la structure primaire de la protéine des cheveux. La quantité d'hélices, conformation fréquente de la structure secondaire des protéines, dans les cheveux de groupes de femmes d'âges différents, a également été analysée par microscopie infrarouge couplée à une résolution multivariée de courbes (RCM). À partir des résultats, une image de la distribution bidimensionnelle des hélices alpha a été générée pour les cheveux prélevés dans chaque groupe d'âge. De plus, une calorimétrie différentielle à balayage haute pression (HPDSC) des cheveux dans l'eau a été effectuée sur les cheveux prélevés dans chaque groupe d'âge pour déterminer la température maximale de l'effet endothermique et l'enthalpie de la dénaturation. RÉSULTATS: Nous avons constaté que la teneur en groupe amino dans les protéines capillaires diminuait et que la kératine de type II, l'une des sous-unités du filament intermédiaire, était plus carbonylée avec l'âge. Les résultats de la RCM ont mis en évidence huit composants distincts, y compris des composants de la structure secondaire des protéines, tels que les hélices α et les feuillets ß. Des images bidimensionnelles des coupes transversales de cheveux ont révélé que la présence d'hélices alpha diminuait avec l'âge. En outre, les données de l'HPDSC ont montré que l'enthalpie associée à la température de dénaturation diminuait également de manière significative avec l'âge. CONCLUSION: Ces résultats suggèrent qu'il existe une corrélation négative entre l'âge et l'intégrité structurelle du segment hélicoïdal dans le filament intermédiaire. Les résultats de cette étude montrent également qu'il existe une corrélation positive entre la dénaturation du cheveu liée à l'âge et les protéines carbonyles.

Prediction of breakage in human hair caused by cyclical extension using infrared spectroscopy coupled with multivariate curve resolution.

We propose a method of prediction for hair breakage induced by change of molecular structure. The changes inside hair by cyclical extension were investigated using infrared (IR) spectroscopy coupled with multivariate curve resolution (MCR) and two-dimensional correlation spectroscopy (2D-COS). In bleached hair, cyclical extension stresses at 5% strain levels were seen to increase the signal of CH2 and that of component primarily derived from C=O at 55 cycles, SO3H signal at 75 cycles, ultimately leading to fiber breakage. The CH2 profile of bleached hair significantly increased at 55 cycles compared to before extension in almost agreement with component profile primarily derived from C=O. This agreement behavior was likely to surface translocation of the lipids such as wax esters and triacylglycerols inside hair caused by cyclical extension. Two-dimensional correlation spectra indicated that SO3H was produced by way of a cystine oxide by cyclical extension. In contrast, only CH2 signal gradually increased without breakage in untreated hair. Thus, the method proposed in this study monitoring three functional groups is expected to have potential application in prediction of hair breakage by cyclical extension such as everyday grooming actions.

A new approach to removing interference of moisture from FTIR spectrum.

An approach is developed to remove the interference of moisture from FTIR spectra. The interference arises from two aspects: the fluctuation on the temperature of the HeNe laser and the fluctuation on the transient concentration of moisture in the light - path of an FTIR spectrometer. The temperature fluctuation on the HeNe laser produces a systematic spectral shift between single-beam sample and background spectra, which often makes spectral subtraction method invalid in removing the interference of moisture. Herein, the Carbo similarity metric (the CAB value) is used to reflect the subtle spectral shift. A database of single-beam background spectra is established based on the concept of big-data and the pigeon-hole theory. The spectral shift is corrected by selecting suitable single-beam background spectra from the database to match with the given single-beam sample spectrum according to the CAB value. The interference caused by the fluctuation of the transient concentration of moisture is removed using a comprehensive 2D-COS method. We apply the approach on two polymeric samples to retrieve high-quality spectra and reliable second derivative spectra without the interference of moisture. The present work provides a new opportunity of obtaining the reliable second derivative spectra in the spectral region masked by moisture.

Different hydration states and passive tumor targeting ability of polyethylene glycol-modified dendrimers with high and low PEG density.

It has been reported that the amount of intermediate water, defined as water molecules loosely bound to a material, is a useful index of the material's bio-inert properties. Polyethylene glycol (PEG) is a well-known biocompatible polymer with a large amount of intermediate water. Many researchers have showed that PEGylated nanoparticles are passively accumulated in tumor tissues owing to their enhanced permeability and retention (EPR) effects. Dendrimers are regularly branched polymers with highly controllable size and structure, which can be exploited as potent drug carriers. In this study, we investigated the tripartite relationship among the PEG density, the hydration state, and the passive tumor targeting property, using PEGylated dendrimers. The fully PEGylated dendrimer, PEG64-den, showed similar hydration behavior to PEG and a passive tumor targeting property. In contrast, the hydration state of the partly PEGylated dendrimer, PEG5-den, was different from that of PEG64-den, and the passive tumor targeting property was not observed. This is the first report to show the hydration state of a drug carrier as well as discuss a relationship between the hydration state and biodistribution.

Understanding phase transition and vibrational mode coupling in ammonium nitrate using 2D correlation Raman spectroscopy.

Ammonium nitrate (AN) is an important component of the chemical industry such as an active ingredient in fertilizers, as an oxidizer in explosive compositions and propellants, and as a blasting agent in civil explosives. Numerous accidents have been reported in the past which concerns its thermal instability and poses a big threat to its processing, transportation, and storage. Despite much literature being reported to understand its thermal instability, a mechanistic view remains unclear. In the present work, we have studied the behavior of AN to temperature change using a mathematical approach called 2D correlation (2D Cos) Raman spectroscopy to provide complete insight into the detailed dynamical nature of the interactions between the species (ionic or molecular) occurring with an increase in temperature. We have analyzed various libration and translational modes of nitrate in the low-frequency region using this mathematical tool. It is observed from 2D maps that the phase transition of AN starts with changes in libration modes followed by various nitrate modes and ammonium modes which further precedes low-frequency translational modes. Further, the 2D correlation could differentiate between modes splitting and shifting based on specific 2D Cos pattern. The changes occurring in the N-O deformation modes, symmetric stretching modes as well as anti-symmetric stretching modes which have been attributed to the weakening of the hetero-ionic coupling between the NH4+ and the NO3- ions could be clearly distinguished in the 2D synchronous and asynchronous plots. Besides, moving window analysis was performed to visualize the transition temperature at which phase change of AN takes place.

Role of interfacial water in determining the interactions of proteins and cells with hydrated materials.

Water molecules play a crucial role in biointerfacial interactions, including protein adsorption and desorption. To understand the role of water in the interaction of proteins and cells at biological interfaces, it is important to compare particular states of hydration water with various physicochemical properties of hydrated biomaterials. In this review, we discuss the fundamental concepts for determining the interactions of proteins and cells with hydrated materials along with selected examples corresponding to our recent studies, including poly(2-methoxyethyl acrylate) (PMEA), PMEA derivatives, and other biomaterials. The states of water were analyzed by differential scanning calorimetry, in situ attenuated total reflection infrared spectroscopy, and surface force measurements. We found that intermediate water which is loosely bound to a biomaterial, is a useful indicator of the bioinertness of material surfaces. This finding on intermediate water provides novel insights and helps develop novel experimental models for understanding protein adsorption in a wide range of materials, such as those used in biomedical applications.

Use of Near-Infrared-Mid-Infrared Dual-Wavelength Spectrometry to Obtain Two-Dimensional Difference Spectra of Sesame Oil as Inactive Drug Ingredient.

The present study has investigated the transformation of sesame oil kept at low temperature during a definite period of time for refinement (called winterization) as an inactive drug ingredient by using two-dimensional difference spectra (2D-DS) analysis of spectra collected using a near-infrared (NIR) and mid-infrared (MIR) dual-wavelength spectrometer (NIR-MIR-DWS). The NIR and MIR spectra were measured nearly simultaneously from samples of sesame oil before and after winterization. The difference spectrum analysis of the obtained NIR-MIR data elucidated that, after the winterization process, the absorbances at peaks attributed to C=O, C=C, and OH groups decrease while the absorbances arising from the main chain (CH2) increase. The result indicated the removal of lignan and the fatty acids with relatively short main chains. Moreover, sesame oil unwinterized was cooled from room temperature to near 1 ℃ and subsequently warmed to room temperature. And the cycle was repeated two times. Real-time monitoring during the cooling and warming processes were carried out using the NIR-MIR-DWS. The prediction results obtained from partial least square calibration model for the temperature suggests that there are subtle differences in the oil composition between the first cooling process and after the warming and cooling cycle. For the more detailed analysis, the 2D-DS method is proposed. The results of the analyses using 2D-DS revealed that the starting point of the transformation is around 15 ℃. It can be estimated that sesame oil is mainly transformed by the first cooling down. Moreover, it was implied that the structure of methylene (CH2) was significantly related to the modifications in sesame oil with temperature change. A series of experimental results elucidated that the winterization of sesame oil removed its impurities and stabilized its conditions. These results are probably the first report on the effect of the winterization process on sesame oil.

Real-time determination and visualization of two independent quantities during a manufacturing process of pharmaceutical tablets by near-infrared hyperspectral imaging combined with multivariate analysis.

During pharmaceutical manufacturing, line-scan hyperspectral imaging enables us to collect several electromagnetic spectra at each pixel in a two-dimensional plane for each tablet. The present study quantitatively determines two independent values of the active pharmaceutical ingredient (API) content in a tablet and the amount of coating on a surface of the same tablet simultaneously; the process is visualized by means of a near-infrared hyperspectral imaging (NIR-HSI) system combined with multivariate data analysis at a typical manufacturing speed of 4,000 tablets per minute. The API content and the amount of coating were controlled to be in the range 80-120% and 0-7 mg, respectively. The results of the cross validation of regression models demonstrated a coefficient of determination (R2) of 0.942, a root-mean-square error of cross validation (RMSECV) of 3.48% for the API content, an R2 of 0.939, and an RMSECV of 0.46 mg for the amount of coating. These results demonstrated that the API content in a tablet as well as the amount of coating on the surface of the same tablet can be simultaneously determined with sufficient accuracy. This technique is practically applicable to process analytical technology in pharmaceutical manufacturing.

Chemometrics and Related Fields in Python.

The Python programing language is becoming a promising tool for data analysis in various fields. However, little attention has been paid to using Python in the field of analytical chemistry, though recent advances in instrumental analysis require robust and reliable data analysis. In order to overcome the difficulty in accurate analysis, multivariate analysis, or chemometrics, has been widely applied to various kinds of data obtained by instrumental analysis. In the present work, the potential usefulness of Python for chemometrics and related fields in chemistry is reviewed. Many practical tools for chemometrics, e.g., principal component analysis (PCA), partial least squares (PLS), support vector machine (SVM), etc., are included in the scikit-learn machine learning (ML) library for Python. Other useful libraries such as pyMCR for multivariate curve resolution (MCR), 2Dpy for two-dimensional correlation spectroscopy (2D-COS), etc. can be obtained from GitHub. For these reasons, a computational environment for chemometrics is easily constructed in Python.

A novel systematic absence of cross peaks-based 2D-COS approach for bilinear data.

A novel approach to use two-dimensional correlation spectroscopy (2D-COS) to analyze bilinear data is proposed. A phenomenon called Systematic Absence of Cross Peaks (SACPs) is observed in a 2D asynchronous spectrum. Two theorems relevant to SACPs have been derived. The SACP-based 2D-COS method has been successfully applied on analyzing bilinear data from mixed samples (including one model system and two real systems). Implicit isolated peaks can be identified and assigned to different components based on characteristic pattern of SACPs even if the time-related profiles of different components are severely overlapped. Based on the results of SACPs, spectra of pure components can be retrieved. Identification of SACPs can still be achieved in the presence of artifacts. Thus, neither noise nor baseline drift can produce significant influence on the results obtained from the approach described in this paper. We have used several well-established chemometric methods, including N-Findr, VCA, and MCR with various initial settings, on two systems that can be successfully solved using the 2D-COS method. The chemometric methods mentioned above cannot provide correct spectra of pure components because of severe problem of rotational ambiguity derived from severe overlapping of the time-related profiles. Only when the information from SACPs in 2D-COS is used as additional constraints in MCR calculation, correct spectra can be obtained. That is to say, the SACP-based 2D-COS method provides intrinsic information which is crucial in the analysis of chromatographic-spectroscopic and analogous data even if the time-related profiles of different components overlap severely.

Novel Method of Constructing Two-Dimensional Correlation Spectroscopy without Subtracting a Reference Spectrum.

In this study, we propose a new approach to generate two-dimension spectra to enhance the intensity of cross peaks relevant to intermolecular interaction. We investigate intermolecular interaction between two solutes (denoted as P and Q, where P has a characteristic peak at XP) dissolved in the same solvent via the near diagonal cross peaks around the coordinate (XP, XP) in a two-dimensional (2D) asynchronous spectrum of generalized spectroscopy. Because of physical constrains in many cases, the variation ranges of the initial concentrations of P or Q must be kept very narrow, leading to very weak cross peak intensities. The weak cross peaks vulnerable to noise bring about difficulty in the investigation of subtle intermolecular interaction. Herein, we propose a new of way constructing a 2D asynchronous spectrum without the subtraction of the average spectrum often used as a reference spectrum. Mathematical analysis and computer simulation demonstrate that the near diagonal cross peaks around the coordinate (XP, XP) in the 2D asynchronous spectrum using the new approach possess two characteristics: (1) they can still reflect an intermolecular interaction reliably; 2) the absolute intensities of the cross peaks are significantly stronger than those generated by the conventional method. We incorporate the novel method with the DAOSD (double asynchronous orthogonal sample design scheme) approach and applied the modified DAOSD approach to study hydrogen bonding behavior in diethyl either/methanol/THF system. The new approach made the weak cross peaks, which are not observable in 2D asynchronous spectrum generated using conventional approach, become observable. The appearance of the cross peak demonstrate that When a small amount of THF is introduced into diethyl solution containing low amount of methanol, THF breaks the methanol-diethyl ether complex and forms methanol-THF complex via new hydrogen bond. This process takes place in spite of the fact that the content of diethyl ether is overwhelmingly larger than that of THF. The above result demonstrates that the new approach described in this article is applicable to enhance intensity of cross peaks in real chemical systems.

Multivariate curve resolution using a combination of mid-infrared and near-infrared spectra for the analysis of isothermal epoxy curing reaction.

Multivariate curve resolution (MCR) was applied to a hetero-spectrally combined dataset consisting of mid-infrared (MIR) and near-infrared (NIR) spectra collected during the isothermal curing reaction of an epoxy resin. An epoxy monomer, bisphenol A diglycidyl ether (BADGE), and a hardening agent, 4,4'-diaminodiphenyl methane (DDM), were used for the reaction. The fundamental modes of the NH and OH stretches were highly overlapped in the MIR region, while their first overtones could be independently identified in the NIR region. The concentration profiles obtained by MCR using the hetero-spectral combination showed good agreement with the results of calculations based on the Beer-Lambert law and the mass balance. The band assignments and absorption sites estimated by the analysis also showed good agreement with the results using two-dimensional (2D) hetero-correlation spectroscopy.

Surface force and vibrational spectroscopic analyses of interfacial water molecules in the vicinity of methoxy-tri(ethylene glycol)-terminated monolayers: mechanisms underlying the effect of lateral packing density on bioinertness.

Unequivocal dependence of bioinertness of self-assembled monolayers of methoxy-tri(ethylene glycol)-terminated alkanethiol (EG3-OMe SAMs) on their packing density has been a mystery for more than two decades. We tackled this long-standing question by performing surface force and surface-enhanced infrared absorption (SEIRA) spectroscopic measurements. Our surface force measurements revealed a physical barrier of interfacial water in the vicinity of the Au-supported EG3-OMe SAM (low packing density), whereas the Ag-supported one (high packing density) did not possess such interfacial water. In addition, the results of SEIRA measurements clearly exhibited that hydrogen bonding states of the interfacial water differ depending on the substrates. We also characterized the bioinertness of these SAMs by protein adsorption tests and adhesion assays of platelet and human umbilical vein endothelial cells. The hydrogen bonding states of the interfacial water and water-induced interaction clearly correlated with the bioinertness of the SAMs, suggesting that the interfacial water plays an important role determining the interaction of the SAMs with biomolecules and cells.

Moving-window two-dimensional heterospectral (MW2DHetero) correlation analysis and its application for the process monitoring of alcoholic fermentation.

The technique of moving-window two-dimensional heterospectral (MW2DHetero) correlation spectroscopy is proposed. This computational method is based on the ideas of perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy and two-dimensional heterospectral correlation analysis. Not only small spectral variations, but also detailed bands assignments were captured using the analysis. This method was applied to near-infrared (NIR) spectra in the 10 000-4000 cm(-1) region and mid-infrared (mid-IR) spectra in the 5000-1200 cm(-1) region, which were simultaneously detected using a dual-region spectrometer. Near-infrared and mid-IR spectra collected during an alcoholic fermentation process using a solution containing glucose and fructose were reported. Slight time differences for the consumption of sugars compared with the production of ethanol were found between 50 and 150 min. It was concluded that these slight time differences are evidence for different consumption times between glucose and fructose during the fermentation process. The result proved a possibility of the selective monitoring of the simultaneous reaction processes between productive and consumptive components.

Developing dissolution testing methodologies for extended-release oral dosage forms with supersaturating properties. Case example: Solid dispersion matrix of indomethacin.

The objective of this study was to develop an in vitro dissolution test method with discrimination ability for an extended-release solid dispersion matrix of a lipophilic drug using the United States Pharmacopeia (USP) Apparatus 4, flow-through cell apparatus. In the open-loop configuration, the sink condition was maintained by manipulating the flow rate of the dissolution medium. To evaluate the testing conditions, the drug release mechanism from an extended-release solid dispersion matrix containing hydrophobic and hydrophilic polymers was investigated. As the hydroxypropyl methylcellulose (HPMC) maintained concentrations of indomethacin higher than the solubility in a dissolution medium, the release of HPMC into the dissolution medium was also quantified using size-exclusion chromatography. We concluded that the USP Apparatus 4 is suitable for application to an in vitro dissolution method for orally administered extended-release solid dispersion matrix formulations containing poorly water-soluble drugs.

Effect of sodium chloride on hydration structures of PMEA and P(MPC-r-BMA).

The hydration structures of two different types of biomaterials, i.e., poly(2-methoxyethyl acrylate) (PMEA) and a random copolymer of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate (P(MPC-r-BMA)), were investigated by means of attenuated total reflection infrared (ATR-IR) spectroscopy. The effects of the addition of sodium chloride to liquid water in contact with the surfaces of the polymer films were examined. The neutral polymer of PMEA was easily dehydrated by NaCl addition, whereas the zwitterionic polymer of P(MPC-r-BMA) was hardly dehydrated. More specifically, nonfreezing water having a strong interaction with the PMEA chain and freezing bound water having an intermediate interaction were hardly dehydrated by contacting with normal saline solution, whereas freezing water having a weak interaction with the PMEA chain was readily dehydrated. In contrast, freezing water in P(MPC-r-BMA) is exchanged for the saline solution contacting with the material surface without dehydration.

Hydrogen-bonds structure in poly(2-hydroxyethyl methacrylate) studied by temperature-dependent infrared spectroscopy.

Hydrogen-bonds structure in poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated by means of temperature-dependent infrared (IR) spectroscopy. Spectral variations involved with the OH…OH and C=O…HO types of hydrogen-bonds were found around the glass transition temperature of 80°C. Hydrogen-bonds among the hydroxyl groups gradually dissociate with increasing temperature. In contrast, discontinuous variation in the carbonyl bands was observed around the glass transition temperature. An association of the C=O…HO type of hydrogen-bond with increasing temperature above the glass transition temperature was revealed. These were concluded from the present study that hydrogen-bonds among the hydroxyl groups in each side chain terminal suppress the main chain mobility in the polymer matrix below the glass transition temperature, while the dissociation of the OH…OH type of hydrogen-bonds induces the association of the C=O…HO type of hydrogen-bond. As a result, the mobility of the main chain is induced by the change in hydrogen-bonds structure at the glass transition temperature.

Thermal behavior of poly(lactic acid)-nanocomposite studied by near-infrared imaging based on roundtrip temperature scan.

The thermal behavior of poly(lactic acid) (PLA) was studied by near-infrared imaging to provide a molecular-level understanding of the physical improvement caused by nanoclay dispersion. A set of PLA samples, each having different nanoclay dispersion, was prepared under varying sonication time. Crystallinity variation of the polymer interacting with the nanoclay particles was analyzed by a roundtrip temperature scan below the melting temperature. Namely, the samples underwent heating and then cooling in the opposite way during the spectral measurement. The discrepancy of the spectral feature between the heating and the cooling indicated the development of the hysteresis associated with the cold crystallization of the PLA lamellae. The generation of the spectral residuals revealed the inner working mechanism of how the polymer structure undergoes variation depending on the presence of the clay particles and their dispersions. The sonication brings substantial dispersion of the nanoclay over the polymer matrix. The nanoclay particles then induce the additional development of the crystalline structure due to the molecular interaction between the PLA and nanoclay arising from the presence of enormous surface area, which in turn induces variation of mechanical strength to the polymer.

Two-step curing reaction of epoxy resin studied by thermal analysis and infrared spectroscopy.

A curing reaction of bisphenol A diglycidyl ether epoxy resin with 4,4'-diaminodicyclohexyl methane hardener was investigated by means of modulated differential scanning calorimetry (MDSC) and infrared (IR) spectroscopy. MDSC observation revealed that the curing process of the resin occurred in two steps. Mid-infrared and near-infrared spectra of the resin were measured as a function of temperature. The obtained spectra were analyzed by perturbation-correlation moving-window two-dimensional correlation spectroscopy (PCMW2D-COS). The first step was revealed as a polymerization reaction among the oxirane group and primary and secondary amine groups, followed by etherification; the second step of the curing process occurred in the vicinity of the gelation point and was characterized by the growth of a three-dimensional cross-linking structure with tertiary amine and etherification of the hydroxyl group.

Near-infrared imaging of water in a polymer electrolyte membrane during a fuel cell operation.

A novel technique of spectroscopic imaging using a near-infrared (NIR) laser sheet beam was developed for visualization of liquid water in a proton-exchange membrane (PEM) sandwiched between two opaque electrodes set in a polymer electrolyte fuel cell (PEFC). In-plane two-dimensional distribution of water in the thin membrane was clearly visualized during the fuel cell operation. Under the condition of fuel feeding into the PEFC without humidification, water was generated by the fuel cell reaction in the whole electrode area. In contrast, under the condition of fuel feeding with humidification, the PEM got wet in the vicinity of a gas flow field locally.