Raman spectral imaging of single cancer cells: probing the impact of sample fixation methods

Raman spectroscopy has proven its potential for the analysis of cell constituents and processes. However, sample preparation methods compatible with clinical practice must be implemented for collection of accurate spectral information. This study aims at assessing, using micro-Raman imaging, the effects of some routinely used fixation methods such as formalin-fixation, formalin-fixation/air drying, cytocentrifugation, and air drying on intracellular spectral information. Data were compared with those acquired from single living cells. In parallel to these spectral information, cell morphological modifications that accompany sample preparation were compared. Spectral images of isolated cells were first analyzed in an unsupervised way using hierarchical cluster analysis (HCA), which allowed delimitation of the cellular compartments. The resulting nuclei cluster centers were compared and revealed at the molecular level that fixation induced changes in spectral information assigned to nucleic acids and proteins. In a second approach, a supervised fitting procedure using model spectra of DNA, RNA, and proteins, chemically extracted from living cells, revealed very small modifications at the level of the localization and quantification of these macromolecules. Finally, HCA and principal components analysis (PCA) performed on individual spectra randomly selected from the nuclear regions showed that formalin-fixation and cytocentrifugation are sample preparation methods that have little impact on the biochemical information as compared to living conditions. Any step involving cell air drying seems to accentuate the spectral deviations from the other preparation methods. It is therefore important in a future context of spectral cytology to take into account these variations.     

IR spectroscopy reveals effect of non-cytotoxic doses of anti-tumour drug on cancer cells

Identifying early cellular events in response to a chemotherapy drug treatment, in particular at low doses that will destroy the highest possible number of cancer cells, is an important issue in patient management. In this study, we employed Fourier transform infrared spectroscopy as a potential tool to access such information. We used as model the non-small cell lung cancer cell line, Calu-1. They were exposed to cytostatic doses (0.1 to 100 nM for 24, 48 and 72 h) of gemcitabine, an anti-tumour drug, currently used in treatment of lung cancer patients. In these conditions, inhibition of cell proliferation ranges from weak (≤5%), to moderate (∼23%), to high (82–95%) without affecting cell viability. Following drug treatment as a function of doses and incubation times, the spectra of cell populations and of individual cells were acquired using a bench-top IR source and a synchrotron infrared microscope. It is demonstrated that spectral cell response to gemcitabine is detectable at sublethal doses and that effects observed on cell populations are similar to those from single cells. Using cluster analysis, spectra could be classified in two main groups: a first group that contains spectra of cells exhibiting a weak or moderate proliferation rate and a second group with spectra from cells presenting a high growth inhibition. These results are promising since they show that effects of subtoxic doses can also be monitored at the single-cell level with the clinical implications that this may have in terms of patient benefit and response to chemotherapy.     

Polymer-modification of an anti-cancer drug: effects on hydrodynamic properties

Coupling of a hydrophobic pharmaceutical agents with the hydrophilic poly(ethylene oxide) chain can make the drug water soluble which is necessary for some applications. The modification of the polymer chain results in a different solution behaviour of the macromolecules compared with the unmodified polymer. There are strong indications that shape and rigidity of the modified molecule differ significantly from the simple chain. Some results from measurements of the self-diffusion coefficient and solution viscosity are discussed. The results are important for further engineering on active drug systems.     

Surface-enhanced Raman scattering imaging of single living lymphocytes with multivariate evaluation

This paper is aimed to show the possibility to determine individual organic compounds introduced into single living cells with surface-enhanced Raman spectroscopy (SERS). Surface enhancement was achieved with gold colloids that were allowed to diffuse into lymphocytes. An introduced analyte, rhodamine 6G, could be imaged together with for example nucleotides and amino acids of the cell. Multivariate evaluation of surface-enhanced Raman images proved to be a powerful tool for the separation of spectral information of various intracellular components. The principal component analysis (PCA) enabled identification of spectra containing different chemical information and separation of the spectral contribution of rhodamine 6G from the complex cellular matrix.     

Imaging the cell wall of living single yeast cells using surface-enhanced Raman spectroscopy

The surface of a living yeast cell (Saccharomyces cerevisiae strain W303-1A) has been labeled with silver (Ag) nanoparticles that can form nanoaggregates which have been shown to have surface-enhanced Raman scattering (SERS) activity. The cell wall of a single living yeast cell has been imaged by use of a Raman microspectroscope. The SERS spectra measured from different Ag nanoaggregates were found to be different. This can be explained on the basis of detailed spectral interpretation. The SERS spectral response originates from mannoproteins which cover the outermost regions of the yeast cell wall. Analysis of SERS spectra from the cell wall and the extracted mannoproteins from the yeast has been performed for the clarification of variation in SERS spectra.     

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.     

Spectral unmixing and clustering algorithms for assessment of single cells by Raman microscopic imaging

A detailed comparison of six multivariate algorithms is presented to analyze and generate Raman microscopic images that consist of a large number of individual spectra. This includes the segmentation algorithms for hierarchical cluster analysis, fuzzy C-means cluster analysis, and k-means cluster analysis and the spectral unmixing techniques for principal component analysis and vertex component analysis (VCA). All algorithms are reviewed and compared. Furthermore, comparisons are made to the new approach N-FINDR. In contrast to the related VCA approach, the used implementation of N-FINDR searches for the original input spectrum from the non-dimension reduced input matrix and sets it as the endmember signature. The algorithms were applied to hyperspectral data from a Raman image of a single cell. This data set was acquired by collecting individual spectra in a raster pattern using a 0.5-??m step size via a commercial Raman microspectrometer. The results were also compared with a fluorescence staining of the cell including its mitochondrial distribution. The ability of each algorithm to extract chemical and spatial information of subcellular components in the cell is discussed together with advantages and disadvantages.     

Multiplexed multicolor Raman imaging of live cells with isotopically modified single walled carbon nanotubes

We show that single walled carbon nanotubes (SWNTs) with different isotope compositions exhibit distinct Raman G-band peaks and can be used for multiplexed multicolor Raman imaging of biological systems. Cancer cells with specific receptors are selectively labeled with three differently "colored" SWNTs conjugated with various targeting ligands including Herceptin (anti-Her2), Erbitux (anti-Her1), and RGD peptide, allowing for multicolor Raman imaging of cells in a multiplexed manner. SWNT Raman signals are highly robust against photobleaching, allowing long-term imaging and tracking. With narrow peak features, SWNT Raman signals are easily differentiated from the autofluorescence background. The SWNT Raman excitation and scattering photons are in the near-infrared region, which is the most transparent optical window for biological systems in vitro and in vivo. Thus, SWNTs are novel Raman tags promising for multiplexed biological detection and imaging.     

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

A quantitative SERS imaging strategy is developed for O-GlcNAcylation mapping of single living cells through a competitive click reaction.     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

In situ Raman imaging of astaxanthin in a single microalgal cell

Raman imaging is shown to be a highly selective and sensitive method of studying in situ and in vivo astaxanthin distribution, concentration and molecular structure in the cyst form of the unicellular microalgae Haematococcus pluvialis.     

Raman spectroscopic identification of single bacterial cells under antibiotic influence

The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

Development of an oxidative dehydrogenation-based fluorescent probe for Cu and its biological imaging in living cells

Based on a boron dipyrromethene (BODIPY) derivative containing an N, O and S tridentate ligand, a Cu²⁺ fluorescent probe BTCu was developed. The detection mechanism was verified as Cu²⁺-promoted oxidative dehydrogenation of an amine moiety, leading to a formation of a fluorescent Cu⁺-Schiff base complex. Free BTCu exhibited a maximum absorption wavelength at 496 nm, and a very weak maximum emission at 511 nm. Upon addition of various metals ions, it showed large fluorescence enhancement toward Cu²⁺ (417-fold in MeCN and 103-fold in MeCN/HEPES solution, respectively) with high selectivity. The detection limits are as low as 1.74 × 10⁻⁸ M and 4.96 × 10⁻⁸ M in the two different solutions, respectively. And BTCu could work in a wide pH range with an extraordinary low pK_a of 1.21 ± 0.06. Using fluorescence microscopy, the probe was shown to be capable of penetrating into living cells and imaging intracellular Cu²⁺ changes.     

Label-free coherent anti-stokes Raman scattering imaging of coexisting lipid domains in single bilayers

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy was used to image lipid domains in single bilayers without any labeling. On the basis of the molecular packing density difference between liquid-disordered (Ld), liquid-ordered (Lo), and gel (So) phases, clear vibrational contrasts were generated between coexisting domains in a single bilayer of DOPC/DPPC (1:1) and DOPC/DPPC/cholesterol (4:4:2). The method reported here can be potentially applied to study phase segregation in live cell membranes which are highly heterogeneous and dynamic.     

Raman spectroscopic analysis of apoptosis of single human gastric cancer cells

A drug (5-FU) was employed to treat the gastric carcinoma cells and induce apoptosis of the cancer cells. Raman spectra obtained from single gastric carcinoma cells and the induced apoptotic cells through scan-excitation mode were used to analyze the effectiveness of the treatment. The major difference of the apoptotic cells from the cancer cells are the reduction in intensities of vibration bands generated by cellular lipids, proteins and nucleic acids. In particular, large intensity reduction in nucleic vibrations at 782, 1092, 1320, 1340, and 1578 cm⁻¹ was observed upon apoptosis of the gastric carcinoma cells. Up to 45% reduction in the magnitude of the 782 cm⁻¹ peak in Raman spectra of the apoptotic cells was observed, which suggests the breakdown of phosphodiester bonds and DNA bases. We showed that the principal components analysis (PCA), a multivariate statistical tool, can be used to distinguish single apoptotic cells and gastric carcinoma cells based on their Raman spectra.     

Hydrogen bonding effects on infrared and Raman spectra of drug molecules

Infrared and Raman spectra of three drug molecules, aspirin, caffeine and ibuprofen, in gas phase and in aqueous solution have been simulated using hybrid density functional theory. The long range solvent effect is modelled by the polarizable continuum model, while the short range hydrogen bonding effects are taken care of by the super-molecular approach with explicit inclusion of water molecules. The calculated spectra are found to compare well with available experimental results. The agreement obtained make grounds for proposing theoretical modeling as a tool for characterizing changes in the bonding environments of drug molecules in terms of particular variations in their IR and Raman spectra.     

Walking nanothermometers: spatiotemporal temperature measurement of transported acidic organelles in single living cells

We fabricated fluorescent nanoparticles which monitor temperature changes without sensitivity to pH (4-10) and ionic strength (0-500 mM). The nanothermometers spontaneously enter living HeLa cells via endocytosis, enclosed in acidic organelles, i.e., endosome/lysosome, and then transported along microtubules in a temperature-dependent manner, working as "walking nanothermometers".     

Fast quantification of water in single living cells by near-infrared microscopy

We have set up a near-infrared microscope using a tuneable diode laser in the range from 1530 to 1570 nm. This spectral range is close to the peak of the water overtone absorption. We used this new microscope to study liver cells, hepatocytes, showing that quantitative information of the intracellular water concentration in living cells can be extracted.