Conventional Raman Microscopy Research Articles

Slit-scanning Raman microscopy: Instrumentation and applications for molecular imaging of cell and tissue

In recent years, Raman microscopy has emerged as a molecular imaging tool for cell and tissue analysis. A key reason for this is the development of techniques that significantly increase imaging speed. In this Tutorial, we introduce slit-scanning Raman microscopy, a Raman imaging technique that achieves imaging speeds more than two orders of magnitude faster than conventional confocal Raman microscopy, and its application to cell and tissue imaging and analysis. Recent advances in Raman imaging, particularly further improvements in imaging speed, sensitivity, specificity, and spatial resolution, are also discussed. In addition, we present the prospects of Raman microscopy as a molecular imaging method to aid in new discoveries in life sciences and the potential of high-speed Raman imaging for clinical applications.

Lithium Insertion into Graphitic Carbon Observed via Operando Kerr-Gated Raman Spectroscopy Enables High State of Charge Diagnostics.

Monitoring the precise lithium inventory of the graphitic carbon electrode within the Li-ion battery, in order to assess cell aging, has remained challenging. Herein, operando electrochemical Kerr-gated Raman spectroscopy measurements on microcrystalline graphite during complete lithium insertion and extraction are reported and compared to conventional continuous-wave Raman microscopy. Suppression of the fluorescence emission signals via use of the Kerr gate enabled the measurement of the Raman graphitic bands of highly lithiated graphite where 0.5 ≤ x ≤ 1 for LixC6. The broad graphitic band initially centered at ca. 1590 cm–1 for Li0.5C6 linearly shifted to ca. 1564 cm–1 with further lithiation to LiC6, thus offering a sensitive diagnostic tool to interrogate high states of charge of graphitic carbon-based negative electrodes.

Surface Plasmon Localization-Based Super-resolved Raman Microscopy.

We investigate label-free measurement of molecular distribution by super-resolved Raman microscopy using surface plasmon (SP) localization. Localized SP was formed with plasmonic nanopost arrays (PNAs) for measurement of the molecular distribution in HeLa cells. Compared with conventional Raman microscopy on gold thin films, PNAs induce a localized near-field, which allows for the enhancement of the peak signal-to-noise ratio by as much as 4.5 dB in the Raman shifts. Super-resolved distributions of aromatic amino acids and lipids (C-C stretching and C-H2 twist mode) as targets in HeLa cells were obtained after image reconstruction. Results show almost 4-fold improvement on average in the lateral precision over conventional diffraction-limited Raman microscopy images. Combined with axial imaging in an evanescent field, the results suggest an improvement in optical resolution due to superlocalized light volume by more than an order of magnitude over that of conventional diffraction-limited Raman microscopy.

Advances in Raman spectroscopy for the non-destructive subsurface analysis of artworks: Micro-SORS

A new analysis modality in Raman microscopy, Micro-Spatially Offset Raman Spectroscopy (micro-SORS) to explore the subsurface of materials in non-destructive and non-invasive way has been developed recently at ICVBC – CNR. Micro-SORS facilitates the investigation of the chemical composition of subsurface, micrometre-scale-thick diffusely scattering layers at depths more than an order of magnitude larger than those accessible with the depth resolving power of conventional confocal Raman microscopy, avoiding need to resort to cross sectional analysis or sampling. In the state of the art paragraph an overall overview of research carried out at CNR-ICVBC in the last five years concerning the development, optimization and application of micro-SORS to Cultural Heritage materials is provided. Additionally, we present new data achieved from the application of micro-SORS to illustrative micro-samples obtained from mediaeval polychrome sculptures of the Parma baptistery and Ferrara cathedral, highlighting further its potential in Cultural Heritage sciences.

Rapid, 3D Chemical Profiling of Individual Atmospheric Aerosols with Stimulated Raman Scattering Microscopy

AbstractVisualizing the 3D chemical profiles of individual aerosols is crucial to understand their formation and aging processes, yet remains technically challenging. Here, the first application of stimulated Raman scattering (SRS) microscopy on 3D chemical imaging of individual aerosols in a nondestructive manner is demonstrated. SRS is capable of mapping chemical components of aerosols at a speed four orders of magnitude faster than conventional spontaneous Raman microscopy. Spatially resolved distributions of nitrates and sulfates reveal the fine structures and different mixing states of atmospheric particles. Moreover, high‐throughput quantifications of chemical compositions and particle size distributions are realized by large‐area imaging and statistical analysis. Its high‐speed and 3D chemical quantification capabilities promise SRS microscopy as a unique tool for studying the properties of single atmospheric particles, and ultimately their impacts on climate and human health.

Detection of low dose of piroxicam polymorph in pharmaceutical tablets by surface-enhanced Raman chemical imaging (SER-CI) and multivariate analysis.

This study demonstrates, for the first time, the ability of surface-enhanced Raman chemical imaging (SER-CI) combined with multivariate analysis to detect low levels (0.1% (w/w)) of a polymorphic form in a pharmaceutical mixture. In the studied formulation, piroxicam was used as a model molecule to develop this approach. Piroxicam is a widely available non-steroidal anti-inflammatory drug, exhibiting an interesting case of polymorphism, with two most commonly observed forms (β and α2). In this work, the SERS spectra of piroxicam polymorphic forms β and α2 are presented. These forms showed clear spectral differences in terms of band position and intensity. From a crystallographic point of view, the difference of exaltation between both forms was correlated with a preferred orientation of crystallites of form α2 making its SERS detection difficult compared to form β. A preferred orientation of the (1k0) crystallographic planes of α2 was demonstrated in samples, not promoting an appropriate molecular orientation onto the metallic surface. Additionally, a semi-quantitative approach using SER-CI combined with chemometric tools was developed enabling to detect crystallites of form β below the detection limit of conventional Raman microscopy. The exaltation of the Raman signal in the presence of silver nanoparticles allowed a higher sensitivity and a reduction of the acquisition time by a factor of 6.

Vibrational Imaging and Quantification of Two-Dimensional Hexagonal Boron Nitride with Stimulated Raman Scattering.

Hexagonal boron nitride (h-BN) is an important member of two-dimensional (2D) materials with a large direct bandgap, and has attracted growing interest in ultraviolet optoelectronics and nanoelectronics. Compared with graphene and graphite, h-BN has weak Raman effect because of the far off-resonance excitation; hence, it is difficult to exploit Raman spectroscopy to characterize important properties of 2D h-BN, such as thickness, doping, and strain effects. Here, we applied stimulated Raman scattering (SRS) to enhance the sensitivity of the E2g Raman mode of h-BN. We showed that SRS microscopy achieves rapid high resolution imaging of h-BN with a pixel dwell time 4 orders of magnitude smaller than conventional spontaneous Raman microscopy. Moreover, the near-perfect linear dependence of signal intensity on h-BN thickness and isotropic polarization dependence allow convenient determination of the flake thickness with SRS imaging. Our results indicated that SRS microscopy provides a promising tool for high-speed quantification of h-BN and holds the potential for vibrational imaging of 2D materials.

Speeding Up the Line-Scan Raman Imaging of Living Cells by Deep Convolutional Neural Network.

Raman imaging is a promising technique that allows the spatial distribution of different components in the sample to be obtained using the molecular fingerprint information on individual species. However, the imaging speed is the bottleneck for the current Raman imaging methods to monitor the dynamic process of living cells. In this paper, we developed an artificial intelligence assisted fast Raman imaging method over the already fast line scan Raman imaging method. The reduced imaging time is realized by widening the slit and laser beam, and scanning the sample with a large scan step. The imaging quality is improved by a data-driven approach to train a deep convolutional neural network, which statistically learns to transform low-resolution images acquired at a high speed into high-resolution ones that previously were only possible with a low imaging speed. Accompanied with the improvement of the image resolution, the deteriorated spectral resolution as a consequence of a wide slit is also restored, thereby the fidelity of the spectral information is retained. The imaging time can be reduced to within 1 min, which is about five times faster than the state-of-the-art line scan Raman imaging techniques without sacrificing spectral and spatial resolution. We then demonstrated the reliability of the current method using fixed cells. We finally used the method to monitor the dynamic evolution process of living cells. Such an imaging speed opens a door to the label-free observation of cellular events with conventional Raman microscopy.

Standoff Raman Detection of Explosive Materials Using a Small Raman Spectroscopy System

In this work a standoff Raman spectroscopy SRS system has been designed, assembled andtested for detecting explosives (Ammonium nitrate, Trinitrotoluene and Urea nitrate) in darklaboratory at 4 m target-telescope distance. The SRS system employs frequency doubled Nd:YAGlaser at 532 nm excitation with laser power of 250 mW and integration time of 2 second. The Cassegrain telescope was coupled to the Ventana Raman spectrometer using a fiber optics cable, and Notch filter is used to reject Rayleigh scattering light. The Raman scattered light is collected by a telescope and then transferred via fiber optic to spectrometer and finally directed into charge coupled device CCD detector. In order to test SRS system, it has been used to detect the Raman spectra of Toxic Industrial Compounds TIC such as acetone, toluene, and carbon tetrachloride. The SRS results were compared with conventional Raman microscopy results using a bench top Bruker SENTERRA Raman instrument.

Fast and reproducible iSERS microscopy of single HER2-positive breast cancer cells using gold nanostars as SERS nanotags.

Speed is often a bottleneck in conventional Raman microscopy on biological specimens. In immuno-Raman microspectroscopy, or for short iSERS microscopy, the acquisition times per pixel have been reduced by more than one order of magnitude over the past decade since its proof of concept. Typically rather high laser power densities are employed with the intention of compensating for the shorter acquisition times, without checking the reproducibility of the results in repeated experiments on the same sample. Here, we systematically analyze this aspect at the single-cell level since it forms the basis of quantification and is very important for reinspection of the same specimen. Specifically, we investigate experimentally the role of the laser power density in conjunction with the acquisition times per pixel in a series of repeated iSERS experiments on the same single cell overexpressing the breast cancer tumor marker human epidermal growth factor receptor 2 (HER2). Confocal iSERS mapping experiments were guided by wide-field fluorescence microscopy for selecting the regions of interest. We demonstrate that the combination of ca. a 1-2 mW laser power (40× objective, NA 0.6), 50 ms acquisition time per pixel and a high EM-CCD signal gain yields highly reproducible iSERS images in a series of four repeated experiments on the same single cell. In contrast, longer acquisition times (0.8 s, no EM gain) and in particular higher laser power (4 mW up to 18 mW) densities lead to non-reproducible iSERS results due to signal degradation.

Surface-enhanced Raman spectroscopy (SERS) in cultural heritage.

Surface-enhanced Raman spectroscopy (SERS) has been increasingly used in the last decade for the identification of organic colourants in works of art. 'Surface-enhancement' is the giant increase in Raman scattering intensity experienced by organic molecules adsorbed on atomically rough metallic surfaces. In the case of organic colourants, which are in general strongly luminescent under laser excitation and therefore cannot usually be identified by conventional Raman microscopy (see AMCTB no. 67), SERS yields a strong and clearly resolved spectrum, allowing for easy identification. Unlike ordinary Raman spectroscopy, SERS often requires sampling, yet the sample size is considerably smaller (from 20 to 100 micrometres) than that required for high-performance liquid chromatography (HPLC), the benchmark technique for organic colourants analysis. For this reason, SERS has seen substantial use in the analysis of works of art such as prints, drawings, paintings, and polychrome sculpture. This technical brief focuses on the practical aspects of SERS and its application to the analysis of cultural heritage material, as well as on its current limitations.

Development of a full micro-scale spatially offset Raman spectroscopy prototype as a portable analytical tool.

We present, for the first time, a portable full micro-Spatially Offset Raman Spectroscopy (micro-SORS) prototype permitting the in situ analysis of thin, highly turbid stratified layers at depths not accessible to conventional Raman microscopy. The technique is suitable for the characterisation of painted layers in panels, canvases and mural paintings, painted statues and decorated objects in cultural heritage or stratified polymers, and biological, catalytic and forensics samples where invasive analysis is undesirable or impossible to perform. The new device is characterised conceptually in polymer and paint layer systems. The provision of portability with full micro-SORS delivers subsurface micro-SORS capability unlocking the non-invasive and non-destructive potential of micro-SORS at its most effective form permitting it to be applied to large and non-portable objects in situ without recourse to removing micro-fragments for laboratory analysis on benchtop Raman microscopes.

Discovering Hidden Painted Images: Subsurface Imaging Using Microscale Spatially Offset Raman Spectroscopy.

We demonstrate for the first time the mapping capability of micro-spatially offset Raman spectroscopy (micro-SORS). The technique enables to form noninvasive images of thin sublayers through highly turbid overlayers. The approach is conceptually demonstrated on recovering overpainted images in situations where conventional Raman microscopy was unable to visualize the sublayer. The specimens mimic real situations encountered in Cultural Heritage that deal, for example, with hidden paintings vandalized with graffiti or covered by superimposed painted layers or whitewash. Additionally, using a letter as a hidden image, we demonstrated the micro-SORS potential to reconstruct also a hidden writing covered, for example, with paper sheets that cannot be easily removed. Potential applications could also include other disciplines such as polymers, biological, catalytic, and forensic sciences where thin, highly turbid layers mask chemically distinct subsurface structures.

Determination of thickness of thin turbid painted over-layers using micro-scale spatially offset Raman spectroscopy.

We present a method for estimating the thickness of thin turbid layers using defocusing micro-spatially offset Raman spectroscopy (micro-SORS). The approach, applicable to highly turbid systems, enables one to predict depths in excess of those accessible with conventional Raman microscopy. The technique can be used, for example, to establish the paint layer thickness on cultural heritage objects, such as panel canvases, mural paintings, painted statues and decorated objects. Other applications include analysis in polymer, biological and biomedical disciplines, catalytic and forensics sciences where highly turbid overlayers are often present and where invasive probing may not be possible or is undesirable. The method comprises two stages: (i) a calibration step for training the method on a well characterized sample set with a known thickness, and (ii) a prediction step where the prediction of layer thickness is carried out non-invasively on samples of unknown thickness of the same chemical and physical make up as the calibration set. An illustrative example of a practical deployment of this method is the analysis of larger areas of paintings. In this case, first, a calibration would be performed on a fragment of painting of a known thickness (e.g. derived from cross-sectional analysis) and subsequently the analysis of thickness across larger areas of painting could then be carried out non-invasively. The performance of the method is compared with that of the more established optical coherence tomography (OCT) technique on identical sample set.This article is part of the themed issue 'Raman spectroscopy in art and archaeology'.

Investigation of SEI Growth on Carbon-Coated ZnFe2O4 Li-Ion Battery Anode Material By I n Situ Raman Microscopy

Lithium-ion batteries (LIBs) are electrochemical devices for energy storage used in portable electronics due to their high energy density, lightweight and flexible design. However, improvements in safety, cost and energy and power density to meet the requirements of automotive industry and large scale storage technology for the wide application of LIBs on these fields.[1, 2] The battery performance is affected by surface layers formed on the electrodes. In particular, the solid electrolyte interphase (SEI) is a protecting surface film formed on the negative electrode in organic-based electrolytes. It consists of inorganic and organic species resulting from electrolyte decomposition.[3, 4] This electron insulating layer allows the transport of lithium ions and it has an impact on the irreversible charge loss, rate capability, cyclability and safety.[3] Therefore, it is essential to understand the processes that occur at the electrode/electrolyte interfaces and the nature of the surface film to improve the efficiency and develop longer-life LIBs. ZnFe2O4 (ZFO) is a non-toxic and cheap material of great interest as anode for LIBs with a high specific theoretical capacity ca. 1000 mAhg-1 due to a conversion-alloying reaction that involves the intake of 9 Li+.[5] An environmental friendly method to encapsulate ZFO nanoparticles within a carbon matrix has been described to avoid the aggregation of particles and loss of electronic conductivity that leads to capacity fading.[5] This work presents in situ Raman measurements of carbon-coated ZnFe2O4(ZFO-C) material during the first discharge/charge cycle.[6] The Raman spectrum of ZFO-C electrode at the open-circuit potential (OCP) only shows the D and G bands characteristic of carbon materials. These peaks weaken during the discharge step suggesting that the lithiation also occurs in the carbon coating. The signal of the D and G bands is recovered at the end of the charge step. The absence of peaks related to ZFO material indicates that the carbon coating remains mechanically intact after the first discharge/charge cycle. New Raman bands are observed within a potential region that coincides with SEI layer formation. These bands have been assigned to poly(ethylene)oxide species and organic lithium alkyl carbonates from electrolyte side reactions. The detection of SEI compounds by conventional Raman microscopy may be the result of “temporary surface enhancement Raman effect” from Zn nanoparticles formed during the conversion reaction of ZnFe2O4 in a similar potential region as SEI formation. The observation of these polymeric species formed on the carbon coating is comparable to shell isolated nanoparticle Raman spectroscopy (SHINERS) reported by Tian and co-workers.[7]

Analytical Capability of Defocused µ-SORS in the Chemical Interrogation of Thin Turbid Painted Layers.

A recently developed micrometer-scale spatially offset Raman spectroscopy (μ-SORS) method provides a new analytical capability for investigating non-destructively the chemical composition of sub-surface, micrometer-scale thickness, diffusely scattering layers at depths beyond the reach of conventional confocal Raman microscopy. Here, we demonstrate experimentally, for the first time, the capability of μ-SORS to determine whether two detected chemical components originate from two separate layers or whether the two components are mixed together in a single layer. Such information is important in a number of areas, including conservation of cultural heritage objects, and is not available, for highly turbid media, from conventional Raman microscopy, where axial (confocal) scanning is not possible due to an inability to facilitate direct imaging within the highly scattering sample. This application constitutes an additional capability for μ-SORS in addition to its basic capacity to determine the overall chemical make-up of layers in a turbid system.

Development of portable defocusing micro-scale spatially offset Raman spectroscopy.

We present, for the first time, portable defocusing micro-Spatially Offset Raman Spectroscopy (micro-SORS). Micro-SORS is a concept permitting the analysis of thin, highly turbid stratified layers beyond the reach of conventional Raman microscopy. The technique is applicable to the analysis of painted layers in cultural heritage (panels, canvases and mural paintings, painted statues and decorated objects in general) as well as in many other areas including polymer, biological and biomedical applications, catalytic and forensics sciences where highly turbid stratified layers are present and where invasive analysis is undesirable or impossible. So far the technique has been demonstrated only on benchtop Raman microscopes precluding the non-invasive analysis of larger samples and samples in situ. The new set-up is characterised conceptually on a range of artificially assembled two-layer systems demonstrating its benefits and performance across several application areas. These included stratified polymer sample, pharmaceutical tablet and layered paint samples. The same samples were also analysed by a high performance (non-portable) benchtop Raman microscope to provide benchmarking against our earlier research. The realisation of the vision of delivering portability to micro-SORS has a transformative potential spanning across multiple disciplines as it fully unlocks, for the first time, the non-invasive and non-destructive aspects of micro-SORS enabling it to be applied also to large and non-portable samples in situ without recourse to removing samples, or their fragments, for laboratory analysis on benchtop Raman microscopes.

Fast Confocal Raman Imaging Using a 2-D Multifocal Array for Parallel Hyperspectral Detection.

We present the development of a novel confocal hyperspectral Raman microscope capable of imaging at speeds up to 100 times faster than conventional point-scan Raman microscopy under high noise conditions. The microscope utilizes scanning galvomirrors to generate a two-dimensional (2-D) multifocal array at the sample plane, generating Raman signals simultaneously at each focus of the array pattern. The signals are combined into a single beam and delivered through a confocal pinhole before being focused through the slit of a spectrometer. To separate the signals from each row of the array, a synchronized scan mirror placed in front of the spectrometer slit positions the Raman signals onto different pixel rows of the detector. We devised an approach to deconvolve the superimposed signals and retrieve the individual spectra at each focal position within a given row. The galvomirrors were programmed to scan different focal arrays following Hadamard encoding patterns. A key feature of the Hadamard detection is the reconstruction of individual spectra with improved signal-to-noise ratio. Using polystyrene beads as test samples, we demonstrated not only that our system images faster than a conventional point-scan method but that it is especially advantageous under noisy conditions, such as when the CCD detector operates at fast read-out rates and high temperatures. This is the first demonstration of multifocal confocal Raman imaging in which parallel spectral detection is implemented along both axes of the CCD detector chip. We envision this novel 2-D multifocal spectral detection technique can be used to develop faster imaging spontaneous Raman microscopes with lower cost detectors.

Wide-field Raman imaging for bone detection in tissue.

Inappropriate bone growth in soft tissue can occur after trauma to a limb and can cause a disruption to the healing process. This is known as Heterotopic Ossification (HO) in which regions in the tissue start to mineralize and form microscopic bone-like structures. These structures continue to calcify and develop into large, non-functional bony masses that cause pain, limit limb movement, and expose the tissue to reoccurring infections; in the case of open wounds this can lead to amputation as a result of a failed wound. Both Magnetic Resonance Imaging (MRI) and X-ray imaging have poor sensitivity and specificity for the detection of HO, thus delaying therapy and leading to poor patient outcomes. We present a low-power, fast (1 frame per second) optical Raman imaging system with a large field of view (1 cm(2)) that can differentiate bone tissue from soft tissue without spectroscopy, this in contrast to conventional Raman microscopy systems. This capability may allow for the development of instrumentation which permits bedside diagnosis of HO.

Monte Carlo simulations of subsurface analysis of painted layers in micro-scale spatially offset Raman spectroscopy.

A recently developed micrometer-scale spatially offset Raman spectroscopy (micro-SORS) method provides a new analytical capability for investigating nondestructively the chemical composition of subsurface, micrometer-scale-thick, diffusely scattering layers at depths beyond the reach of conventional confocal Raman microscopy. Here we provide, for the first time, the theoretical foundations for the micro-SORS defocusing concept based on Monte Carlo simulations. Specifically, we investigate a defocusing variant of micro-SORS that we used in our recent proof-of-concept study in conditions involving thin, diffusely scattering layers on top of an extended, diffusely scattering substrate. This configuration is pertinent, for example, for the subsurface analysis of painted layers in cultural heritage studies. The depth of the origin of Raman signal and the relative micro-SORS enhancement of the sublayer signals reached are studied as a function of layer thickness, sample photon transport length, and absorption. The model predicts that sublayer enhancement initially rapidly increases with increasing defocusing, ultimately reaching a plateau. The magnitude of the enhancement was found to be larger for thicker layers. The simulations also indicate that the penetration depths of micro-SORS can be between one and two orders of magnitude larger than those reached using conventional confocal Raman microscopy. The model provides a deeper insight into the underlying Raman photon migration mechanisms permitting the more effective optimization of experimental conditions for specific sample parameters.