RESUMO
Mitochondrial activity is a widely used criterion to judge the metabolic condition of a living specimen. Numerous methods have been developed for related analyses, including the detection of O2 consumption, trans-membrane potential, and ATP production. In this study, we demonstrate that the redox state of cytochromes can serve as a sensitive mitochondrial activity indicator in glutamate-stressed neuronal cells. Mitochondrial dysfunction was detected by Raman imaging as early as 30 min after glutamate-stress induction. By comparing this result with other commonly used mitochondrial function assays, we found Raman imaging has a similar sensitivity to ATP production and trans-membrane potential assays. Other viability tests, such as MTT assay and ROS production tests, showed a slower response than our method. A thorough understanding of cytochrome dynamics with our new method will help establish Raman spectroscopy as a competitive clinical diagnosis tool for neurodegenerative diseases involving mitochondrial dysfunction.
Assuntos
Citocromos/química , Citocromos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/diagnóstico , Animais , Linhagem Celular , Ácido Glutâmico , Camundongos , Doenças Mitocondriais/induzido quimicamente , Oxirredução , Análise Espectral Raman/métodosRESUMO
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
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
BACKGROUND: Lipid/carbohydrate content and ratio are extremely important when engineering algal cells for liquid biofuel production. However, conventional methods for such determination and quantification are not only destructive and tedious, but also energy consuming and environment unfriendly. In this study, we first demonstrate that Raman spectroscopy is a clean, fast, and accurate method to simultaneously quantify the lipid/carbohydrate content and ratio in living microalgal cells. RESULTS: The quantification results of both lipids and carbohydrates obtained by Raman spectroscopy showed a linear correspondence with that obtained by conventional methods, indicating Raman can provide a similar accuracy to conventional methods, with a significantly shorter detection time. Furthermore, the subcellular resolution of Raman spectroscopy enabled not only the concentration mapping of lipid/carbohydrate content in single living cells, but also the evaluation of standard deviation between the biomass accumulation levels of individual algal cells. CONCLUSIONS: In this study, we first demonstrate that Raman spectroscopy can be used for starch quantification in addition to lipid quantification in algal cells. Due to the easiness and non-destructive nature of Raman spectroscopy, it makes a perfect tool for the further study of starch-lipid shift mechanism.
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
Raman spectral imaging is gaining more and more attention in biological studies because of its label-free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information. The dual-polarization Raman images of a HeLa cell.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Imagem Molecular/métodos , Análise Espectral Raman/métodos , Sobrevivência Celular , Citocromos c/metabolismo , Células HeLa , HumanosRESUMO
Using Raman spectral imaging, we visualized the cell state transition during differentiation and constructed hypothetical potential landscapes for attractors of cellular states on a state space composed of parameters related to the shape of the Raman spectra. As models of differentiation, we used the myogenic C2C12 cell line and mouse embryonic stem cells. Raman spectral imaging can validate the amounts and locations of multiple cellular components that describe the cell state such as proteins, nucleic acids, and lipids; thus, it can report the state of a single cell. Herein, we visualized the cell state transition during differentiation using Raman spectral imaging of cell nuclei in combination with principal component analysis. During differentiation, cell populations with a seemingly homogeneous cell state before differentiation showed heterogeneity at the early stage of differentiation. At later differentiation stages, the cells returned to a homogeneous cell state that was different from the undifferentiated state. Thus, Raman spectral imaging enables us to illustrate the disappearance and reappearance of an attractor in a differentiation landscape, where cells stochastically fluctuate between states at the early stage of differentiation.
Assuntos
Diferenciação Celular , Análise Espectral Raman , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Camundongos , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/metabolismoRESUMO
In the last couple of decades, the spatial resolution in optical microscopy has increased to unprecedented levels by exploiting the fluorescence properties of the probe. At about the same time, Raman imaging techniques have emerged as a way to image inherent chemical information in a sample without using fluorescent probes. However, in many applications, the achievable resolution is limited to about half the wavelength of excitation light. Here we report the use of structured illumination to increase the spatial resolution of label-free spontaneous Raman microscopy, generating highly detailed spatial contrast from the ensemble of molecular information in the sample. Using structured line illumination in slit-scanning Raman microscopy, we demonstrate a marked improvement in spatial resolution and show the applicability to a range of samples, including both biological and inorganic chemical component mapping. This technique is expected to contribute towards greater understanding of chemical component distributions in organic and inorganic materials.
RESUMO
Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that ß-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.
Assuntos
Calcificação Fisiológica/fisiologia , Microscopia/métodos , Osteoblastos/citologia , Osteoblastos/fisiologia , Análise Espectral Raman/métodos , Imagem com Lapso de Tempo/métodos , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Citocromos c/metabolismo , Camundongos , beta Caroteno/metabolismoRESUMO
System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states.
Assuntos
Diferenciação Celular/fisiologia , Análise Espectral Raman/métodos , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , CamundongosRESUMO
The 1602 cm(-1) Raman signature, which we call the "Raman spectroscopic signature of life" in yeasts, is a marker Raman band for cell metabolic activity. Despite the established fact that its intensity sensitively reflects the metabolic status of the cell, its molecular origin remained unclear. In this work, we propose ergosterol as the major contributor of the 1602 cm(-1) Raman signature. The theoretical isotope shift calculation for ergosterol agreed with previous observations. Furthermore, experiments showed that the Raman spectrum of ergosterol corresponds very well with the depleting spectral component in yeast that behaves together with the 1602 cm(-1) signature when the cells are under stress. This work implies that the 1602 cm(-1) Raman signature could serve as an intrinsic ergosterol marker in yeasts for the study of sterol metabolism in vivo and in a label-free manner, which could not be done by any other techniques at the current stage.
Assuntos
Ergosterol/química , Saccharomyces cerevisiae/citologia , Análise Espectral Raman , Clorofórmio/química , Mitocôndrias/químicaRESUMO
HEM1 gene encodes δ-aminolevulinate synthase that is required for haem synthesis. It is an essential gene for yeast survival. The Raman spectra of HEM1 knockout (hem1Δ) yeast cells lacks a Raman band at 1602 cm(-1) that has been shown to reflect cell metabolic activity. This result suggests that the molecule giving rise to the"Raman spectroscopic signature of life" is closely related to haem functions in the cell. High amount of squalene is also observed in the hem1Δ strain, which is another new discovery of this study.