一种酵母细胞生长现象的实时单细胞拉曼光谱观察

用拉曼镊子观察单个即发活性干酵母(Saccharomy cescerevisiae)细胞在2.0%葡萄糖溶液中的活化过程,收集其拉曼光谱。结果发现,在某一批次的产品中,酵母细胞的1364cm-1峰强度随着细胞的活化而显著增加,531cm-1、652cm-1、1053cm-1等源自葡萄糖或葡萄糖基的信号峰也会随细胞的生长而增强,随后增强的还有1432cm-1、1448cm-1、1561cm-1等源自脂类物质的峰,而源自蛋白质及脂类的1000cm-1、1445cm-1、1655cm-1等峰的信号强度基本不变,酵母细胞代谢活跃的标志峰1603cm-1也基本不变。该批次产品中,10次实验有7次观察到上述现象,而在别的批次产品中并没有观察到该现象。用单细胞拉曼光谱实时记录了这一特殊的生长现象。     

Single cell Raman spectroscopy for cell sorting and imaging

Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free technology, allowing in vivo and multiple parameter analysis of individual living cells. A single cell Raman spectrum usually contains more than 1000 Raman bands which provide rich and intrinsic information of the cell (e.g. nucleic acids, protein, carbohydrates and lipids), reflecting cellular genotypes, phenotypes and physiological states. A Raman spectrum serves as a molecular 'fingerprint' of a single cell, making it possible to differentiate various cells including bacterial, protistan and animal cells without prior knowledge of the cells. However, a key drawback of SCRS is the fact that spontaneous Raman signals are naturally weak; this review discusses recent research progress in significantly enhancing and improving the signal of spontaneous Raman spectroscopy, including resonance Raman spectroscopy (RRS), coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman spectroscopy (SRS) and surface enhanced Raman scattering (SERS). This review focuses on the biotechnological development and the associated applications of SCRS, including Raman activated cell sorting (RACS) and Raman imaging and mapping.     

Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.     

Single-cell ICP-MS to address the role of trace elements at a cellular level

The heterogeneity properties shown by cells or unicellular organisms have led to the development of analytical methods at the single-cell level. In this sense, considering the importance of trace elements in these biological systems, the inductively coupled plasma mass spectrometer (ICP-MS) configured for analyzing single cell has presented a high potential to assess the evaluation of elements in cells. Moreover, advances in instrumentation, such as coupling laser ablation to the tandem configuration (ICP-MS/MS), or alternative mass analyzers (ICP-SFMS and ICP-TOFMS), brought significant benefits, including sensitivity improvement, high-resolution imaging, and the cell fingerprint. From this perspective, the single-cell ICP-MS has been widely reported in studies involving many fields, from oncology to environmental research. Hence, it has contributed to finding important results, such as elucidating nanoparticle toxicity at the cellular level and vaccine development. Therefore, in this review, the theory of single-cell ICP-MS analysis is explored, and the applications in this field are pointed out. In addition, the instrumentation advances for single-cell ICP-MS are addressed.     

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

Near-Real-Time Analysis of the Phenotypic Responses of Escherichia coli to 1-Butanol Exposure Using Raman Spectroscopy

Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.     

An intracellular silver deposition method for targeted detection and chemical analysis of uncultured microorganisms

The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (¹³C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.     

Optically-controlled platforms for transfection and single- and sub-cellular surgery

Improving the resolution of biological research to the single-cell or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredictable failures or mutations in individual cells can lead to serious downstream conditions across the whole organism. The traditional tools of biochemistry struggle to observe such processes: the vast majority are based upon ensemble approaches analysing the properties of bulk populations, which means that details of individual constituents is lost. What are required, then, are tools with the precision and resolution to probe and dissect cells at the single-micron scale: the scale of the individual organelles and structures that control their function. In this review, we highlight the use of highly-focused laser beams to create systems which provide precise control and specificity at the single-cell or even single-micron level. The intense focal points generated can directly interact with cells and cell membranes, which in conjunction with related modalities such as optical trapping provide a broad platform for the development of single-cell and sub-cellular surgery approaches. These highly tuneable tools have been demonstrated to deliver or remove material from cells of interest, and they can simultaneously excite fluorescent probes for imaging purposes or plasmonic structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics research.     

Recent advances of single-cell RNA sequencing technology in mesenchymal stem cell research

Mesenchymal stem cells (MSCs) are multipotent stromal cells with great potential for clinical applications. However, little is known about their cell heterogeneity at a single-cell resolution, which severely impedes the development of MSC therapy. In this review, we focus on advances in the identification of novel surface markers and functional subpopulations of MSCs made by single-cell RNA sequencing and discuss their participation in the pathophysiology of stem cells and related diseases. The challenges and future directions of single-cell RNA sequencing in MSCs are also addressed in this review.     

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry(SCMC) combines features of traditional flow cytometry(i.e.,fluorescence-activated cell sorting) with mass spectrometry,making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms.In this study,we optimized SCMC to analyze hemocytes of the Drosophila innate immune system.We used metal-conjugated antibodies(against cell surface antigens H2,H3,H18,L1,L4,and P1,and intracellular antigens 3 A5 and L2) and anti-IgM(against cell surface antigen L6) to detect the levels of antigens,while anti-GFP was used to detect crystal cells in the immune-induced samples.We investigated the antigen expression profile of single cells and hemocyte populations in naive states,in immune-induced states,in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase(hop~(Tum)) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 [1(3)mbn~1],as well as in stem cell maintenance-defective hdc~(Δ84) mutant larvae.Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes,plasmatocytes,and crystal cells,and delineated the unique immunophenotype of Drosophila mutants.We have identified subpopulations of L2~+/P1~+and L2~+/L4~+/P1~+ transitional phenotype cells in the tumorous strains l(3)mbn~1 and hop~(Tum),respectively,and a subpopulation of L4~+/P1~+ cells upon immune induction.Our results demonstrated for the first time that SCMC,combined with multidimensional bioinformatic analysis,represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.     

拉曼光谱分析技术在细胞生物学研究中的应用进展

细胞是生物体结构和功能的基本单位,自被发现以来新的研究方法不断涌现。单细胞拉曼光谱能提供细胞内核酸、蛋白质、脂质含量等大量信息,可在不损伤细胞的条件下实时动态地监测细胞分子结构变化,亦可获得细胞的“分子指纹”,具有敏感性高、实时检测、活样品不需固定或染色、不损伤细胞等众多特点。近年来国内外研究者将拉曼光谱应用于细胞药物处理、细胞水平疾病诊断、单细胞生命活动监测、亚细胞结构等研究,取得了不同程度的进展。随着研究的深入,拉曼光谱分析技术必将在干细胞,癌症研究、细胞分选、药物筛选等领域大有作为。     

Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.     

Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm⁻¹, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis.     

Cell Optical Density and Molecular Composition Revealed by Simultaneous Multimodal Label-Free Imaging

We show how Raman imaging can be combined with independent but simultaneous phase measurements of unlabeled cells, with the resulting data providing information on how the light is retarded and/or scattered by molecules in the cell. We then show, for the first time to our knowledge, how the chemistry of the cell highlighted in the Raman information is related to the cell quantitative phase information revealed in digital holographic microscopy by quantifying how the two sets of spatial information are correlated. The results show that such a multimodal implementation is highly useful for the convenience of having video rate imaging of the cell during the entire Raman measurement time, allowing us to observe how the cell changes during Raman acquisition. More importantly, it also shows that the two sets of label-free data, which result from different scattering mechanisms, are complementary and can be used to interpret the composition and dynamics of the cell, where each mode supplies label-free information not available from the other mode.     

Cell Optical Density and Molecular Composition Revealed by Simultaneous Multimodal Label-Free Imaging

We show how Raman imaging can be combined with independent but simultaneous phase measurements of unlabeled cells, with the resulting data providing information on how the light is retarded and/or scattered by molecules in the cell. We then show, for the first time to our knowledge, how the chemistry of the cell highlighted in the Raman information is related to the cell quantitative phase information revealed in digital holographic microscopy by quantifying how the two sets of spatial information are correlated. The results show that such a multimodal implementation is highly useful for the convenience of having video rate imaging of the cell during the entire Raman measurement time, allowing us to observe how the cell changes during Raman acquisition. More importantly, it also shows that the two sets of label-free data, which result from different scattering mechanisms, are complementary and can be used to interpret the composition and dynamics of the cell, where each mode supplies label-free information not available from the other mode.