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.

Single-molecule tracking of Nanog and Oct4 in living mouse embryonic stem cells uncovers a feedback mechanism of pluripotency maintenance.

Nanog and Oct4 are core transcription factors that form part of a gene regulatory network to regulate hundreds of target genes for pluripotency maintenance in mouse embryonic stem cells (ESCs). To understand their function in the pluripotency maintenance, we visualised and quantified the dynamics of single molecules of Nanog and Oct4 in a mouse ESCs during pluripotency loss. Interestingly, Nanog interacted longer with its target loci upon reduced expression or at the onset of differentiation, suggesting a feedback mechanism to maintain the pluripotent state. The expression level and interaction time of Nanog and Oct4 correlate with their fluctuation and interaction frequency, respectively, which in turn depend on the ESC differentiation status. The DNA viscoelasticity near the Oct4 target locus remained flexible during differentiation, supporting its role either in chromatin opening or a preferred binding to uncondensed chromatin regions. Based on these results, we propose a new negative feedback mechanism for pluripotency maintenance via the DNA condensation state-dependent interplay of Nanog and Oct4.

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.

Application of the Dynamical Network Biomarker Theory to Raman Spectra.

The dynamical network biomarker (DNB) theory detects the early warning signals of state transitions utilizing fluctuations in and correlations between variables in complex systems. Although the DNB theory has been applied to gene expression in several diseases, destructive testing by microarrays is a critical issue. Therefore, other biological information obtained by non-destructive testing is desirable; one such piece of information is Raman spectra measured by Raman spectroscopy. Raman spectroscopy is a powerful tool in life sciences and many other fields that enable the label-free non-invasive imaging of live cells and tissues along with detailed molecular fingerprints. Naïve and activated T cells have recently been successfully distinguished from each other using Raman spectroscopy without labeling. In the present study, we applied the DNB theory to Raman spectra of T cell activation as a model case. The dataset consisted of Raman spectra of the T cell activation process observed at 0 (naïve T cells), 2, 6, 12, 24 and 48 h (fully activated T cells). In the DNB analysis, the F-test and hierarchical clustering were used to detect the transition state and identify DNB Raman shifts. We successfully detected the transition state at 6 h and related DNB Raman shifts during the T cell activation process. The present results suggest novel applications of the DNB theory to Raman spectra ranging from fundamental research on cellular mechanisms to clinical examinations.

Recent Advances in Raman Spectral Imaging in Cell Diagnosis and Gene Expression Prediction.

Normal and tumor regions within cancer tissue can be distinguished using various methods, such as histological analysis, tumor marker testing, X-ray imaging, or magnetic resonance imaging. Recently, new discrimination methods utilizing the Raman spectra of tissues have been developed and put into practical use. Because Raman spectral microscopy is a non-destructive and non-labeling method, it is potentially compatible for use in the operating room. In this review, we focus on the basics of Raman spectroscopy and Raman imaging in live cells and cell type discrimination, as these form the bases for current Raman scattering-based cancer diagnosis. We also review recent attempts to estimate the gene expression profile from the Raman spectrum of living cells using simple machine learning. Considering recent advances in machine learning techniques, we speculate that cancer type discrimination using Raman spectroscopy will be possible in the near future.

Lattice-patterned collagen fibers and their dynamics in axolotl skin regeneration.

The morphology of collagen-producing cells and the structure of produced collagen in the dermis have not been well-described. This lack of insights has been a serious obstacle in the evaluation of skin regeneration. We succeeded in visualizing collagen-producing cells and produced collagen using the axolotl skin, which is highly transparent. The visualized dermal collagen had a lattice-like structure. The collagen-producing fibroblasts consistently possessed the lattice-patterned filopodia along with the lattice-patterned collagen network. The dynamics of this lattice-like structure were also verified in the skin regeneration process of axolotls, and it was found that the correct lattice-like structure was not reorganized after simple skin wounding but was reorganized in the presence of nerves. These findings are not only fundamental insights in dermatology but also valuable insights into the mechanism of skin regeneration.

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells.

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 µm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail-tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.

Valosin-containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations.

Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.

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.

Pressure-induced changes on the morphology and gene expression in mammalian cells.

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper.

Glycine insertion modulates the fluorescence properties of Aequorea victoria green fluorescent protein and its variants in their ambient environment.

The green fluorescent protein (GFP) derived from Pacific Ocean jellyfish is an essential tool in biology. GFP-solvent interactions can modulate the fluorescent property of GFP. We previously reported that glycine insertion is an effective mutation in the yellow variant of GFP, yellow fluorescent protein (YFP). Glycine insertion into one of the ß-strands comprising the barrel structure distorts its structure, allowing water molecules to invade near the chromophore, enhancing hydrostatic pressure or solution hydrophobicity sensitivity. However, the underlying mechanism of how glycine insertion imparts environmental sensitivity to YFP has not been elucidated yet. To unveil the relationship between fluorescence and ß-strand distortion, we investigated the effects of glycine insertion on the dependence of the optical properties of GFP variants named enhanced-GFP (eGFP) and its yellow (eYFP) and cyan (eCFP) variants with respect to pH, temperature, pressure, and hydrophobicity. Our results showed that the quantum yield decreased depending on the number of inserted glycines in all variants, and the dependence on pH, temperature, pressure, and hydrophobicity was altered, indicating the invasion of water molecules into the ß-barrel. Peak shifts in the emission spectrum were observed in glycine-inserted eGFP, suggesting a change of the electric state in the excited chromophore. A comparative investigation of the spectral shift among variants under different conditions demonstrated that glycine insertion rearranged the hydrogen bond network between His148 and the chromophore. The present results provide important insights for further understanding the fluorescence mechanism in GFPs and suggest that glycine insertion could be a potent approach for investigating the relationship between water molecules and the intra-protein chromophore.

Activation probability of a single naïve T cell upon TCR ligation is controlled by T cells interacting with the same antigen-presenting cell.

Accurate recognition of antigens by specific T cells is crucial for adaptive immunity to work properly. The activation of a T-cell antigen-specific response by an antigen-presenting cell (APC) has not been clearly measured at a single T-cell level. It is also unknown whether the cell-extrinsic environment alters antigen recognition by a T cell. To measure the activation probability of a single T cell by an APC, we performed a single-cell live imaging assay and found that the activation probability changes depending not only on the antigens but also on the interactions of other T cells with the APC. We found that the specific reactivity of single naïve T cells was poor. However, their antigen-specific reactivity increased drastically when attached to an APC interacting with activated T cells. Activation of T cells was suppressed when regulatory T cells interacted with the APC. These findings suggest that although the ability of APCs to activate an antigen-specific naïve T cell is low at a single-cell level, the surrounding environment of APCs improves the specificity of the bulk response.

Quantitation of Cell Membrane Permeability of Cyclic Peptides by Single-Cell Cytoplasm Mass Spectrometry.

Cyclic peptides (CPs) have attracted attention as next-generation drugs because they possess both cell-permeable potential as small molecules and specific affinity similar to antibodies. As intracellular molecules are important targets of CPs, quantitation of the intracellular retention and transmembrane permeability of CPs is necessary for drug development. However, permeated CPs within cells cannot be directly assessed by conventional permeability assays using methods such as artificial membranes and cell monolayers. Here, we propose a new approach using single-cell cytoplasm mass spectrometry (SCC-MS). After cells were incubated with CPs, the cytoplasm was directly collected from a single cell using a microneedle followed by nanoelectrospray ionization mass spectrometry detection of the CPs. The height of the CP peak was plotted against time and fitted with a simple function, y = a(1 - e-bx), to calculate the apparent permeability coefficient (Papp) for both the influx and efflux directions. MCF-7 cells were selected as model cancer cells and cultured with cyclosporin A (CsA) and its demethylated analogs (dmCsA-1, -2, and -3) as model CPs. Papp values (10-6 cm/s) obtained from cells incubated with 50 µM CPs ranged from 0.017 to 0.121 for influx and 0.20 to 1.48 for efflux. The higher efflux ratio was possibly caused by efflux transporters such as P-glycoprotein, a well-known receptor of CsA. The equilibrated intracellular concentration of CPs was estimated to be as low as 4.1-6.8 µM, which showed good consistency with the high efflux ratio. SCC-MS is promising as a reliable permeability assay for next-generation CP-based pharmaceuticals.

Analysis of the stability of 70 housekeeping genes during iPS reprogramming.

Studies on induced pluripotent stem (iPS) cells highly rely on the investigation of their gene expression which requires normalization by housekeeping genes. Whether the housekeeping genes are stable during the iPS reprogramming, a transition of cell state known to be associated with profound changes, has been overlooked. In this study we analyzed the expression patterns of the most comprehensive list to date of housekeeping genes during iPS reprogramming of a mouse neural stem cell line N31. Our results show that housekeeping genes' expression fluctuates significantly during the iPS reprogramming. Clustering analysis shows that ribosomal genes' expression is rising, while the expression of cell-specific genes, such as vimentin (Vim) or elastin (Eln), is decreasing. To ensure the robustness of the obtained data, we performed a correlative analysis of the genes. Overall, all 70 genes analyzed changed the expression more than two-fold during the reprogramming. The scale of this analysis, that takes into account 70 previously known and newly suggested genes, allowed us to choose the most stable of all genes. We highlight the fact of fluctuation of housekeeping genes during iPS reprogramming, and propose that, to ensure robustness of qPCR experiments in iPS cells, housekeeping genes should be used together in combination, and with a prior testing in a specific line used in each study. We suggest that the longest splice variants of Rpl13a, Rplp1 and Rps18 can be used as a starting point for such initial testing as the most stable candidates.

Following Embryonic Stem Cells, Their Differentiated Progeny, and Cell-State Changes During iPS Reprogramming by Raman Spectroscopy.

Monitoring cell-state transition in pluripotent cells is invaluable for application and basic research. In this study, we demonstrate the pertinence of noninvasive, label-free Raman spectroscopy to monitor and characterize the cell-state transition of mouse stem cells undergoing reprogramming. Using an isogenic cell line of mouse stem cells, reprogramming from neuronal cells was performed, and we showcase a comparative analysis of living single-cell spectral data of the original stem cells, their neuronal progenitors, and reprogrammed cells. Neural network, regression models, and ratiometric analyses were used to discriminate the cell states and extract several important biomarkers specific to differentiation or reprogramming. Our results indicated that the Raman spectrum allowed us to build a low-dimensional space allowing us to monitor and characterize the dynamics of cell-state transition at a single-cell level, scattered in heterogeneous populations. The ability of monitoring pluripotency by Raman spectroscopy and distinguishing differences between ES and reprogrammed cells is also discussed.

[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.

Multiplexed 129Xe HyperCEST MRI Detection of Genetically Reconstituted Bacterial Protein Nanoparticles in Human Cancer Cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4 and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive, and genetically encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

An improved fluorescent protein-based expression reporter system that utilizes bioluminescence resonance energy transfer and peptide-assisted complementation.

Fluorescent protein-based reporter systems are used to track gene expression in cells. Here, we propose a modified bioluminescence resonance energy transfer (BRET) reporter as a maturation-less reporter that utilizes a peptide-assisted complementation strategy. Using effective dimerized peptides obtained from library-versus-library screening with more than 4000 candidates, rapid activation of the reporter was achieved.

Linking substrate and nucleus via actin cytoskeleton in pluripotency maintenance of mouse embryonic stem cells.

Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process.

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.