RESUMO
Raman scattering microscopy is becoming one of the hot topics in analytical microscopy as a tool for analyzing advanced nanomaterials, such as biomolecules in a live cell for the study of cellular dynamics, semiconductor devices for characterizing strain distribution and contamination, and nanocarbons and nano-2D materials. In this paper, we review the recent progress in the development of Raman scattering microscopy from the viewpoint of spatial resolution and scattering efficiency. To overcome the extremely small cross section of Raman scattering, we discuss three approaches for the enhancement of scattering efficiency and show that the scattering enhancement synergistically increases the spatial resolution. We discuss the mechanisms of tip-enhanced Raman scattering, deep-UV resonant Raman scattering, and coherent nonlinear Raman scattering for micro- and nanoscope applications. The combinations of these three approaches are also shown as nanometer-resolution Raman scattering microscopy. The critical issues of the structures, materials, and reproducibility of tips and three-dimensionality for TERS; photodegradation for resonant Raman scattering; and laser availability for coherent nonlinear Raman scattering are also discussed.
RESUMO
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.
Assuntos
Eletricidade , Luz , Microscopia de Polarização/métodos , Diagnóstico por ImagemRESUMO
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.
RESUMO
Singularity biology is a scientific field that targets drastic state changes in multicellular systems, aiming to discover the key cells that induce the state change and investigate the mechanisms behind them. To achieve this goal, we developed a trans-scale optical imaging system (trans-scale scope), that is capable of capturing both macroscale changes across the entire system and the micro-scale behavior of individual cells, surpassing the cell observation capabilities of traditional microscopes. We developed two units of the trans-scale scope, named AMATERAS-1 and -2, which demonstrated the ability to observe multicellular systems consisting of over one million cells in a single field of view with sub-cellular resolution. This flagship instrument has been used to observe the dynamics of various cell species, with the advantage of being able to observe a large number of cells, allowing the detection and analysis of rare events and cells such as leader cells in multicellular pattern formation and cells that spontaneously initiate calcium waves. In this paper, we present the design concept of AMATERAS, the optical configuration, and several examples of observations, and demonstrate how the strength-in-numbers works in life sciences.
RESUMO
Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.
Assuntos
Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Mioblastos Esqueléticos/fisiologia , Animais , Linhagem Celular , CamundongosRESUMO
We have demonstrated subnanometric stabilization of tip-enhanced optical microscopy under ambient condition. Time-dependent thermal drift of a plasmonic metallic tip was optically sensed at subnanometer scale, and was compensated in real-time. In addition, mechanically induced displacement of the tip, which usually occurs when the amount of tip-applied force varies, was also compensated in situ. The stabilization of tip-enhanced optical microscopy enables us to perform long-time and robust measurement without any degradation of optical signal, resulting in true nanometric optical imaging with high reproducibility and high precision. The technique presented is applicable for AFM-based nanoindentation with subnanometric precision.
Assuntos
Aumento da Imagem/instrumentação , Microscopia de Varredura por Sonda/instrumentação , Nanopartículas/ultraestrutura , Nanotecnologia/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
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.
Assuntos
Análise Espectral Raman , Humanos , Biomarcadores/metabolismo , Análise Espectral Raman/métodos , Progressão da DoençaRESUMO
We have demonstrated the higher yield dimerization of single-crystalline Ag nanocubes through preciously controlled face-selective functionalization. With the achievement of the higher yield dimerization, we could thus observe some interesting optical properties of the dimer. Both experimental and theoretical studies revealed that the 50-nm-diameter Ag nanocubes dimers with a ca. 3.3 nm gap at their junction exhibited two plasmon peaks centered at 446 nm and 600 nm, which contributed to transverse and longitudinal plasmon resonances, respectively. Elctromagnetic calculations based on the FDTD method clearly showed that a greater enhancement of the local field occurred, with an average amplitude of the electric field of 22, at the fractal space between the aggregated Ag nanocubes when the dimer was illuminated under longitudinally polarized light.
Assuntos
Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Prata/química , Ressonância de Plasmônio de Superfície/métodos , Luz , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Refratometria , Espalhamento de Radiação , Propriedades de SuperfícieRESUMO
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.
RESUMO
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.
Assuntos
Citoesqueleto de Actina/patologia , Diferenciação Celular , Núcleo Celular/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas/citologia , alfa Catenina/metabolismo , Quinases Associadas a rho/biossínteseRESUMO
To be able to predict antibiotic resistance in bacteria from fast label-free microscopic observations would benefit a broad range of applications in the biological and biomedical fields. Here, we demonstrate the utility of label-free Raman spectroscopy in monitoring the type of resistance and the mode of action of acquired resistance in a bacterial population of Escherichia coli, in the absence of antibiotics. Our findings are reproducible. Moreover, we identified spectral regions that best predicted the modes of action and explored whether the Raman signatures could be linked to the genetic basis of acquired resistance. Spectral peak intensities significantly correlated (False Discovery Rate, p < 0.05) with the gene expression of some genes contributing to antibiotic resistance genes. These results suggest that the acquisition of antibiotic resistance leads to broad metabolic effects reflected through Raman spectral signatures and gene expression changes, hinting at a possible relation between these two layers of complementary information.
RESUMO
Machine learning-based cell classifiers use cell images to automate cell-type discrimination, which is increasingly becoming beneficial in biological studies and biomedical applications. Brightfield or fluorescence images are generally employed as the classifier input variables. We propose to use Raman spectral images and a method to extract features from these spatial patterns and explore the value of this information for cell discrimination. Raman images provide information regarding distribution of chemical compounds of the considered biological entity. Since each spectral wavelength can be used to reconstruct the distribution of a given compound, spectral images provide multiple channels of information, each representing a different pattern, in contrast to brightfield and fluorescence images. Using a dataset of single living cells, we demonstrate that the spatial information can be ranked by a Fisher discriminant score, and that the top-ranked features can accurately classify cell types. This method is compared with the conventional Raman spectral analysis. We also propose to combine the information from whole spectral analyses and selected spatial features and show that this yields higher classification accuracy. This method provides the basis for a novel and systematic analysis of cell-type investigation using Raman spectral imaging, which may benefit several studies and biomedical applications.
Assuntos
Aprendizado de Máquina , Algoritmos , Animais , Linhagem Celular , Camundongos , Análise de Componente Principal , Análise Espectral RamanRESUMO
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.
Assuntos
Cinesinas , Microtúbulos , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/farmacologia , Animais , Transporte Biológico Ativo , Chlorocebus aethiops , Cães , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Cinesinas/química , Cinesinas/metabolismo , Células Madin Darby de Rim Canino , Microtúbulos/química , Microtúbulos/metabolismo , Células VeroRESUMO
Our current understanding of molecular biology provides a clear picture of how the genome, transcriptome and proteome regulate each other, but how the chemical environment of the cell plays a role in cellular regulation remains much to be studied. Here we show an imaging method using hybrid fluorescence-Raman microscopy that measures the chemical micro-environment associated with protein expression patterns in a living cell. Simultaneous detection of fluorescence and Raman signals, realised by spectrally separating the two modes through the single photon anti-Stokes fluorescence emission of fluorescent proteins, enables the accurate correlation of the chemical fingerprint of a specimen to its physiological state. Subsequent experiments revealed the slight chemical differences that enabled the chemical profiling of mouse embryonic stem cells with and without Oct4 expression. Furthermore, using the fluorescent probe as localisation guide, we successfully analysed the detailed chemical content of cell nucleus and Golgi body. The technique can be further applied to a wide range of biomedical studies for the better understanding of chemical events during biological processes.
Assuntos
Metabolômica , Microscopia de Fluorescência , Proteômica , Análise Espectral Raman , Animais , Biomarcadores , Expressão Gênica , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metabolômica/métodos , Camundongos , Imagem Molecular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteômica/métodos , Proteínas Recombinantes de Fusão , Células-Tronco/metabolismoRESUMO
Scientists are always on the lookout for new modalities of information which could reveal new biological features that are useful for deciphering the complexity of biological systems. Here, we introduce Raman spectroscopy as a prime candidate for ecology and evolution. To encourage the integration of this microscopy technique in the field of ecology and evolution, it is crucial to discuss first how Raman spectroscopy fits within the conceptual, technical and pragmatic considerations of ecology and evolution. In this paper, we show that the spectral information holds reliable indicators of intra- and interspecies variations, which can be related to the environment, selective pressures and fitness. Moreover, we show how the technical and pragmatic aspects of this modality (non-destructive, non-labelling, speed, relative low cost, etc.) enable it to be combined with more conventional methodologies. With this paper, we hope to open new avenues of research and extend the scope of available methodologies used in ecology and evolution.
Assuntos
Evolução Biológica , Ecologia/métodos , Análise Espectral Raman/métodos , Animais , Ecossistema , Especificidade da EspécieRESUMO
Over the past decades many researchers have made major contributions towards the development of genetically encoded (GE) fluorescent sensors derived from fluorescent proteins. GE sensors are now used to study biological phenomena by facilitating the measurement of biochemical behaviors at various scales, ranging from single molecules to single cells or even whole animals. Here, we review the historical development of GE fluorescent sensors and report on their current status. We specifically focus on the development strategies of the GE sensors used for measuring pH, ion concentrations (e.g., chloride and calcium), redox indicators, membrane potential, temperature, pressure, and molecular crowding. We demonstrate that these fluroescent protein-based sensors have a shared history of concepts and development strategies, and we highlight the most original concepts used to date. We believe that the understanding and application of these various concepts will pave the road for the development of future GE sensors and lead to new breakthroughs in bioimaging.
RESUMO
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.
RESUMO
The acquired immune system, mainly composed of T and B lymphocytes, plays a key role in protecting the host from infection. It is important and technically challenging to identify cell types and their activation status in living and intact immune cells, without staining or killing the cells. Using Raman spectroscopy, we succeeded in discriminating between living T cells and B cells, and visualized the activation status of living T cells without labeling. Although the Raman spectra of T cells and B cells were similar, they could be distinguished by discriminant analysis of the principal components. Raman spectra of activated T cells with anti-CD3 and anti-CD28 antibodies largely differed compared to that of naïve T cells, enabling the prediction of T cell activation status at a single cell level. Our analysis revealed that the spectra of individual T cells gradually change from the pattern of naïve T cells to that of activated T cells during the first 24 h of activation, indicating that changes in Raman spectra reflect slow changes rather than rapid changes in cell state during activation. Our results indicate that the Raman spectrum enables the detection of dynamic changes in individual cell state scattered in a heterogeneous population.
Assuntos
Linfócitos B/imunologia , Análise Espectral Raman/métodos , Linfócitos T/imunologia , Animais , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fatores de TempoRESUMO
Fluorescent proteins have been widely used in biology because of their compatibility and varied applications in living specimens. Fluorescent proteins are often undesirably sensitive to intracellular conditions such as pH and ion concentration, generating considerable issues at times. However, harnessing these intrinsic sensitivities can help develop functional probes. In this study, we found that the fluorescence of yellow fluorescent protein (YFP) depends on the protein concentration in the solution and that this dependence can be enhanced by adding a glycine residue in to the YFP; we applied this finding to construct an intracellular protein-crowding sensor. A Förster resonance energy transfer (FRET) pair, involving a cyan fluorescent protein (CFP) insensitive to protein concentration and a glycine-inserted YFP, works as a genetically encoded probe to evaluate intracellular crowding. By measuring the fluorescence of the present FRET probe, we were able to detect dynamic changes in protein crowding in living cells.