Optical evaluation of internal damage to human hair based on second near-infrared window polarization microscopy.

OBJECTIVE: Hair beauty treatments glorify human life. As a side effect, there is a risk of deteriorating the health of the hair. Optically polarized microscopy has been used for many decades to evaluate hair conditions owing to its ease of use and low operating costs. However, the low biopermeability of light hinders the observation of detailed structures inside hair. The aim of this study is to establish an evaluation technique of internal damages in a hair by utilizing a near-infrared (NIR) light with a wavelength of 1000-1600 nm, called "second NIR window". METHODS: We built a laser scanning transmission microscope system with an indium gallium arsenide detector, a 1064 nm laser source, and optical circular polarization to visualize the anisotropy characterization of keratin fibres in hair. Samples of Asian black hair before and after bleaching, after permanent-waving, after lithium bromide (LiBr) treatment, and after heating was observed. Some parameters reflecting intra-hair damage were quantitatively compared with the parameters in digitally recorded images with analytical developments. RESULTS: The light transmittance of black hair was dramatically improved by utilizing the second NIR window. Numerical analysis of circular polarization in hair quantified the internal damage in chemically or thermally treated hair and found two different types of damage. The present method enabled quantitative evaluation of the condition changes in the cortex; for example, a decrease in circular polarizability by LiBr treatment and restoration by replacing the LiBr solution with water. In addition, black speckles were observed after the heat treatment. Longer heating and wetting times increased the appearance probability and size of the speckles. According to quantitative analyses, the emergence of black spots was independent of polarizability changes, indicating that they were not pores. CONCLUSION: Circular polarization microscopy based on near-infrared optics in the second NIR window provides an effective evaluation method for quantifying intra-hair damage caused by cosmetic treatments. The present method provides noninvasive, easy, and inexpensive hair evaluation and has potential as a gold standard in hair care research/medical fields.

OBJECTIF: les soins capillaires glorifient la vie humaine. Comme effet secondaire, il existe un risque de détérioration de la santé du cheveu. La microscopie en lumière polarisée est utilisée depuis de nombreuses décennies pour évaluer la santé capillaire en raison de sa facilité d'utilisation et de son faible coût d'exploitation. Cependant, la faible bioperméabilité de la lumière empêche l'observation des structures détaillées à l'intérieur du cheveu. Pour résoudre ce problème, cette étude tente d'établir une technique d'évaluation des atteintes internes d'un cheveu en utilisant une lumière proche infrarouge (NIR) d'une longueur d'onde de 1000 à 1600 nm, appelée « deuxième fenêtre NIR ¼. MÉTHODES: nous avons construit un système de microscope de transmission à balayage laser équipé d'un capteur indium gallium arsenide, d'une source laser de 1064 nm et d'une polarisation circulaire optique pour visualiser la caractérisation de l'anisotropie des fibres de kératine dans les cheveux. Des échantillons de cheveux noirs asiatiques ont subi un traitement avant et après la décoloration, l'ondulation permanente, le bromure de lithium (LiBr) et la chaleur. Certains paramètres reflétant les dommages intra­cheveu ont été comparés quantitativement aux paramètres des images enregistrées numériquement avec des développements analytiques. RÉSULTATS: la transmission de la lumière des cheveux noirs a été considérablement améliorée en utilisant la deuxième fenêtre NIR. L'analyse numérique de la polarisation circulaire des cheveux a quantifié les dommages internes des cheveux traités chimiquement ou thermiquement et a mis en évidence deux types de dommages différents. La présente méthode a permis d'évaluer quantitativement les changements de condition dans le cortex; par exemple, une diminution de la polarisation circulaire par le traitement par LiBr et la restauration en remplaçant la solution LiBr par de l'eau. En outre, des taches noires ont été observées après le traitement thermique. Des temps de chauffage et de mouillage plus longs ont augmenté la fréquence d'apparition et la taille des taches. D'après des analyses quantitatives, l'émergence de points noirs était indépendante des changements de polarisation, indiquant qu'il ne s'agissait pas de pores. CONCLUSION: La microscopie par polarisation circulaire basée sur l'optique proche infrarouge dans la deuxième fenêtre NIR fournit une méthode d'évaluation efficace pour quantifier les dommages intra­cheveu causés par les traitements cosmétiques. La présente méthode fournit une évaluation des cheveux non invasive, facile et peu coûteuse et a un potentiel de référence dans la recherche sur les soins capillaires/les domaines médicaux.

Improvement in image quality via the pseudo confocal effect in multidirectional digital scanned laser light-sheet microscopy.

Multidirectional digital scanned laser light-sheet microscopy (mDSLM) cannot be used with the current pseudo confocal system to reduce blurring and background signals. The multiline scanning for light-sheet illumination and the simple image construction proposed in this study are alternative to the pseudo confocal system. We investigate the effectiveness of our pseudo confocal method combined with mDSLM on artificial phantoms and biological samples. The results indicate that image quality from mDSLM can be improved by the confocal effect; their combination is effective and can be applied to biological investigations.

Full control of polarization state with a pair of electro-optic modulators for polarization-resolved optical microscopy: erratum.

In our previous paper [Appl. Opt.55, 1082 (2016)APOPAI0003-693510.1364/AO.55.001082], we presented a methodology for full control of a polarization state using a pair of electro-optic modulators. In this erratum, we correct errors in Eqs. (9b) and (9c) in the original paper.

Full control of polarization state with a pair of electro-optic modulators for polarization-resolved optical microscopy.

Full and arbitrary control of polarization states of light using two independent electro-optic modulators is presented. The mechanism of the controllability is theoretically described using the Jones vector and matrix, and the polarization state change with control parameters is geometrically illustrated in the Stokes parameter space. Our theoretical framework involves possible distortions of the polarization state due to optical elements between the polarization controller and measurement point and presents a mechanism for pre-compensating the polarization distortion. The theory's validity and controllability of the polarization state are experimentally demonstrated with a test optical setup using a dichroic mirror as a polarization distorter. The inevitable intensity variation during polarization sweeps and a strategy for pre- and post-compensation of the variations are discussed. The technique's applicability to bioimaging is also discussed.

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.

Non-centrosymmetric coordination polymer with a highly hindered octahedral copper center bridged by mandelate.

A novel chiral coordination polymer, [Cu(C(6)H(5)CH(OH)COO)(µ-C(6)H(5)CH(OH)COO)] (1-L and 1-D), was synthesized through a reaction of copper acetate with L-mandelic acid at room temperature. Although previously reported copper mandelate prepared by hydrothermal reaction was a centrosymmetric coordination polymer because of the racemization of mandelic acid, the current coordination polymer shows noncentrosymmetry and a completely different structure from that previously reported. The X-ray crystallography for 1-L revealed that the copper center of the compound showed a highly distorted octahedral structure bridged by a chiral mandelate ligand in the unusual coordination mode to construct a one-dimensional (1D) zigzag chain structure. These 1D chains interdigitated each other to give a layered structure as a result of the formation of multiple aromatic interactions and hydrogen bonds between hydroxyl and carboxylate moieties at mandelate ligands. The coordination polymer 1-L belongs to the noncentrosymmetric space group of C2 to show piezoelectric properties and second harmonic generation (SHG) activity.

[Application of scattering microscopy for evaluation of iPS cell and its differentiated cells].

Various artificial cells and artificial tissues can be generated from induced pluripotent stem cells (iPS cells). There is now an urgent need to standardize the quality evaluation and management of iPS cells. Recently, artificial intelligence (AI) technology such as machine learning is providing evaluation method for the quality of iPS cells and iPS cell-derived somatic cells based on optical microscopy. Light, which is the principle of optical microscopy, has an interesting and important feature. There are various kinds of interaction between light and molecule, and the scattered light includes internal information of the molecule. Raman scattering inheres all the vibration mode of molecular bonds composing a molecule, and second harmonic generation (SHG) light, which is one of second-order non-linear scattering light, is derived from electric polarizations in the molecule, in other words, carries structural information within the protein. While states of a cell are usually defined by protein/gene expression patterns, we have proposed to apply Raman spectra for cellular fingerprinting as an alternative for identifying the cell state, and now succeeded in predicting gene-expression of antibiotic resistant bacteria in combination with machine learning technology. Meanwhile, SHG microscopy has been used to visualize fiber structures in living specimens, such as collagen, and microtubules as a label-free modality. By utilizing the feature that SHG senses protein structure change, we developed a new method to measure actomyosin activity in cardiac cells. The most important advantage of the use of the scattering light is their non-labeling and non-invasive capability.

Second harmonic generation polarization microscopy as a tool for protein structure analysis.

Cryo-electron microscopy and X-ray crystallography have been the major tools of protein structure analysis for decades and will certainly continue to be essential in the future. Moreover, nuclear magnetic resonance or Förster resonance energy transfer can measure structural dynamics. Here, we propose to add optical second-harmonic generation (SHG), which is a nonlinear optical scattering process sensitive to molecular structures in illuminated materials, to the tool-kit of structural analysis methodologies. SHG can be expected to probe the structural changes of proteins in the physiological condition, and thus link protein structure and biological function. We demonstrate that a conformational change as well as its dynamics in protein macromolecular assemblies can be detected by means of SHG polarization measurement. To prove the capability of SHG polarization measurement with regard to protein structure analysis, we developed an SHG polarization microscope to analyze microtubules in solution. The difference in conformation between microtubules with different binding molecules was successfully observed as polarization dependence of SHG intensity. We also succeeded in capturing the temporal variation of structure in a photo-switchable protein crystal in both activation and inactivation processes. These results illustrate the potential of this method for protein structure analysis in physiological solutions at room temperature without any labeling.

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.

Kinesin-binding-triggered conformation switching of microtubules contributes to polarized transport.

Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

Simultaneous nano-tracking of multiple motor proteins via spectral discrimination of quantum dots.

Simultaneous nanometric tracking of multiple motor proteins was achieved by combining multicolor fluorescent labeling of target proteins and imaging spectroscopy, revealing dynamic behaviors of multiple motor proteins at the sub-diffraction-limit scale. Using quantum dot probes of distinct colors, we experimentally verified the localization precision to be a few nanometers at temporal resolution of 30 ms or faster. One-dimensional processive movement of two heads of a single myosin molecule and multiple myosin molecules was successfully traced. Furthermore, the system was modified for two-dimensional measurement and applied to tracking of multiple myosin molecules. Our approach is useful for investigating cooperative movement of proteins in supramolecular nanomachinery.