Two-dimensional Raman Imaging Research Articles

Multiplex Raman imaging of organelles in endothelial cells

Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Two-dimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells (ECs) was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.

Spatially resolved Raman spectroscopy of defects, strains, and strain fluctuations in domain structures of monolayer graphene

We report spatially resolved Raman scattering results of polycrystalline monolayer graphene films to study the effects of defects, strains, and strain fluctuations on the electrical performance of graphene. Two-dimensional Raman images of the integrated intensities of the G and D peaks (IG and ID) were used to identify the graphene domain boundaries. The domain boundaries were also identified using Raman images of ID/IG and I2D/IG ratios and 2D spectral widths. Interestingly, the ID maps showed that the defects within individual domains significantly increased for the graphene with large domain size. The correlation analysis between the G and 2D peak energies showed that biaxial tensile strain was more developed in the graphene with large domain size than in the graphene with small domain size. Furthermore, spatial variations in the spectral widths of the 2D peaks over the graphene layer showed that strain fluctuations were more pronounced in the graphene with large domain size. It was observed that the mobility (sheet resistance) was decreased (increased) for the graphene with large domain size. The degradation of the electrical transport properties of the graphene with large domain size is mainly due to the defects, tensile strains, and local strain fluctuations within the individual domains.

Confocal raman microspectral imaging of ex vivo human spinal cord tissue

Confocal Raman microspectral imaging (CRMI) provides a versatile tool to illustrate the biochemical nature and structure of biological tissue without introducing any external labels. In this work, a precise correlation was established between the biochemical profile and histological architecture of ex vivo human spinal cord tissue by using CRMI with 633nm excitation. After precisely linking the spectral features to the chemical constituents, much information about the molecular composition of both gray and white matter were revealed. Two-dimensional Raman images were generated by integrating the intensities of the characteristic Raman bands in the area of the intermediate column and ventral horn. K-mean cluster analysis was further applied to visualize the underlying morphological basis of spinal cord tissue by chemical component types and their distribution pattern. Lipid-rich white matter could be visually distinguished from gray matter considering a CH2 bending/scissoring band at 1445cm−1 and an amide III band at 1250cm−1. Meanwhile, the formation and distribution pattern of perineuronal nets (PNNs) in the scanning area was validated by the integration of saccharides (617cm−1) and amide III bands. Moreover, the heme profile indicated a higher degree of vascularization in gray matter. All of the results obtained testified to the possibility that gray matter could be more susceptible to spinal cord injury (SCI) because of capillary network distribution and glycosaminoglycans (GAGs) aggregation. These findings are important for interpreting the morphological specificity of human spinal cord tissue, and also for studying the molecular basis of SCI.

Orientational Ordering of Carotenoids in Myelin Membranes Resolved by Polarized Raman Microspectroscopy

We study orientational ordering of membrane compounds in the myelinated nerve fiber by means of polarized Raman microspectroscopy. The theory of orientational distribution functions was adapted to live-cell measurements. The obtained orientational distribution functions of carotenoids and lipid acyl chain clearly indicated a predominantly radial-like orientation in membranes of the myelin. Two-dimensional Raman images, made under optimal polarization of incident laser beam, corroborated the proposed carotenoid orientation within the bilayer. Experimental data suggested the tilted orientation of both carotenoid polyenic and lipid acyl chains. The values of maximum tilt angles were similar, with possible implication of carotenoid-induced ordering effect on lipid acyl chains, and hence change of myelin membrane properties. This study stages carotenoids of the nerve as possible mediators of excitation and leverages underlying activity-dependent membrane reordering.

Spectroscopic investigations on thin adhesive layers in multi-material laminates.

Three different spectroscopic approaches, Raman linescans, Raman imaging, and attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) imaging were evaluated for the visualization of the thin adhesive layers (3-6 μm) present in polymeric photovoltaic backsheets. The cross-sections of the multilayer laminates in the original, weathered, and artificially aged samples were investigated spectroscopically in order to describe the impact of the environmental factors on the evenness and thickness of the adhesive layers. All three methods were found to be suitable tools to detect and visualize these thin layers within the original and aged polymeric laminates. However, as the adhesive layer is not very uniform in thickness and partly disintegrates upon weathering and/or artificial aging, Raman linescans yield only qualitative information and do not allow for an estimation of the layer thickness. Upon increasing the measuring area by moving from one-dimensional linescans to two-dimensional Raman images, a much better result could be achieved. Even though a longer measuring time has to be taken into account, the information on the uniformity and evenness of the adhesive layer obtainable using the imaging technique is much more comprehensive. Although Raman spectroscopy is known to have the superior lateral resolution as compared with ATR FT-IR spectroscopy, the adhesive layers of the samples used within this study (layer thickness 3-6 μm) could also be detected and visualized by applying the ATR FT-IR spectroscopic imaging method. However, the analysis of the images was quite a demanding task, as the thickness of the adhesive layer was in the region of the resolution limit of this method. The information obtained for the impact of artificial aging and weathering on the adhesive layer obtained using Raman imaging and ATR FT-IR imaging was in good accordance.

Micro-Raman spectroscopic visualization of lattice vibrations and strain in He^+- implanted single-crystal LiNbO_3

Scanning micro-Raman spectroscopy has been utilized to image and investigate strain in He+-implanted congruent LiNbO3 samples. By using abruptly patterned implanted samples, we show that the spatial two-dimensional mapping of the Raman spectral peaks can be used to image the strain distribution and determine its absolute magnitude. We demonstrate that both short- and long-range length-scale in-plane and out-of-plane strain and stress states can be determined using the secular equations of phonon-deformation-potential theory. We also show that two-dimensional Raman imaging can be used to visualize the relaxation of strain in the crystal during low-temperature annealing.

Characterization of nanoprobe uptake in single cells: spatial and temporal tracking via SERS labeling and modulation of surface charge

A critical aspect for use of nanoprobes in biomedical research and clinical applications involves fundamental spatial and temporal characterization of their uptake and distribution in cells. Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate nanoprobes in single cells using surface-enhanced Raman scattering detection. To study the efficiency of cellular uptake, silver nanoparticles functionalized with three different positive-, negative-, and neutrally charged Raman labels were co-incubated with cell cultures and internalized via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake. These results indicate that the functionalized nanoparticle construct has potential for sensing and delivery in single living cells and that use of surface-enhanced Raman scattering for tracking and detection is a practical and advantageous alternative to traditional fluorescence methods. From the Clinical Editor Cell labeling and tracking methods are commonly required in biomedical research. This paper presents specific functionalized nanoparticle constructs with potential for sensing and delivery in single living cells. The use of surface-enhanced Raman scattering enables tracking and detection of these cells as a practical alternative to traditional fluorescence methods.

Two-dimensional Raman imaging for periodic domain structures in laser-patterned ferroelastic β′-(Sm,Gd)2(MoO4)3 crystal lines in glass

Abstract Ferroelastic β′-(Sm,Gd)2(MoO4)3, (SGMO), crystal lines are patterned on the surface of Sm2O3–Gd2O3–MoO3–B2O3 glasses by continuous-wave Yb:YVO4 laser irradiations (wavelength: 1080 nm, power: 1.2 W, scanning speeds: 4–7 μm/s), and the structure of periodic domains self-organized in the crystal lines is examined from two-dimensional micro-Raman scattering spectrum imaging. It is found that the periodicity in the domain structures with different refractive indices changes depending on the laser scanning speed, i.e., on the growth rate of SGMO crystals. It is proposed that the arrangements of (MoO4)2− tetrahedra giving the antisymmetric Mo–O stretching vibrations in SGMO crystals changes along the crystal growth direction (i.e., along the line) gradually and periodically due to spontaneous strains. The present study suggests that the appearance of periodic domain structures and second harmonic intensities is an intrinsic feature in the laser patterning of ferroelastic β′-(Sm,Gd)2(MoO4)3 crystals.

Microscopic Raman Line-Imaging with Principal Component Analysis

Reconstructed Raman images of the distribution of polystyrene and polyethylene were obtained by line-imaging, with the use of univariate and multivariate processing of the spectral data. Multiple sets of microscopic Raman spectral line-images were acquired with line-focused illumination, a motorized translation stage to move the sample perpendicular to the illumination line, and a holographic imaging spectrograph equipped with a 2D detector. The line-imaged raw spectral data were processed with the use of both a simple univariate method (single-band integration) and a more sophisticated multivariate method (principal component analysis with eigenvector rotation) to generate two-dimensional Raman images representing spatial distribution of the polymers. In the spectral range employed (900–1700 cm−1), suitable bands could be found for univariate processing of the polystyrene and polyethylene images. The principal component analysis method gave equivalent separation of the images, but only if the entire spectral window was employed to generate the eigenvectors.

Flame images from pointwise Raman data

Over the past two decades, Raman scattering has been developed as a uniquely useful tool in combustion and gasdynamic research. For flows that are not prohibitively luminous or particle-laden, it can provide composition and temperature data in real time. It has the capability to view physical situations in a “snapshot” fashion—as in single-shot data accumulation to build histograms that can approach probability density functions, or in the direct acquisition of linear or two-dimensional Raman images—and possesses the capability to observe the difference between thermodynamic equilibrium and molecular internal mode populations that are out of equilibrium. Here, we illustrate its applicability to experimental problems for which mean flame structural data are desired. We show here results on the use of pointwise Raman data to give average spatial maps for zones of a turbulent diffusion flame.