3D Raman Imaging Research Articles

Fourier Raman light field microscopy based on surface-enhanced Raman scattering.

Raman scattering, as a vibrational spectrum that carries material information, has no photobleaching that enables long-duration imaging. Raman spectra have very narrow emission peaks, and multiplex Raman imaging can be achieved by using different Raman scattering peak signals. These advantages make Raman imaging widely used in biology, cytology, and medicine, which has a wider range of application scenarios. However, obtaining a three-dimensional (3D) Raman image requires scanning for tens of minutes to several hours at present. Therefore, a fast non-scanning 3D Raman imaging method is greatly needed. In this article, we propose a Fourier Raman light field microscopy based on surface-enhanced Raman scattering (sers-FRLFM). Using flower-like gap-enhanced Raman nanoparticles (F-GERNs) to enhance Raman scattering signals, a Fourier-configured light field microscope (LFM) is capable of recording complete four-dimensional Raman field information in a single frame, facilitating the 3D reconstruction of the Raman image without generating reconstruction artifacts at the native object plan. Moreover, F-GERNs can mark specific locations and have the potential to become a new tracing method to achieve specific imaging. This imaging method has great potential in the 3D real-time Raman imaging of cells, microorganisms, and tissues with the lateral resolution of 2.40 µm and an axial resolution of 4.02 µm.

High-speed and wide-field 3D Raman imaging using flower-like gap-enhanced Raman tags

Raman imaging technology plays a pivotal role in biomedical research, with the pursuit of higher resolution, faster imaging speed, and a larger field of view consistently driving the development of Raman imaging systems. In this work, we propose a high-speed and wide-field three-dimensional (3D) Raman imaging method based on light field microscopy and flower-like gap-enhanced Raman tags (F-GERTs). F-GERTs with enhanced Raman signals and high stability are used to label microbial and cell samples, avoiding the problem of photobleaching. The micro-lens array and scientific camera are incorporated to capture light field Raman images containing spatial information of the sample in a single snapshot acquisition process. The 3D reconstruction algorithm can complete the processing of the light field image within 1 min, reconstructing the 3D Raman image of the sample and the precisely slice structure at different depths. This high-speed and wide-field Raman imaging technology has great potential in the field of real-time 3D Raman imaging of microorganisms and cells.

Hybrid Polypyrrole and Polydopamine Nanosheets for Precise Raman/Photoacoustic Imaging and Photothermal Therapy.

The development of near-infrared light responsive conductive polymers provides a useful theranostic platform for malignant tumors by maximizing spatial resolution with deep tissue penetration for diagnosis and photothermal therapy. Herein, the self-assembly of ultrathin 2D polypyrrole nanosheets utilizing dopamine as a capping agent and a monolayer of octadecylamine as a template is demonstrated. The 2D polypyrrole-polydopamine nanostructure has tunable size distribution which shows strong absorption in the first and second near-infrared windows, enabling photoacoustic imaging and photothermal therapy. The hybrid double-layer is demonstrated to increase Raman intensity for 3D Raman imaging (up to two orders of magnitude enhancement and spatial resolution up to 1µm). The acidic environment drives reversible doping of polypyrrole, which can be detected by Raman spectroscopy. The combined properties of the nanosheets can substantially enhance performance in dual-mode Raman and photoacoustic guided photothermal therapy, as shown by the 69% light to heat conversion efficiency and higher cytotoxicity against cancer spheroids. These pH-responsive features highlight the potential of 2D conductive polymers for applications in accurate, highly efficient theranostics.

The impact of 3D Raman imaging and HR-TEM analysis on the observed changes in the orientation of the crystallites in 3D flower-like nanostructured layers

The presented study aims to explain the formation phenomena of titanium dioxide flower_like forms on Ti foil. TiO2 nanoforms were obtained by chemical oxidation using H2O2 and further crystallization at 600 °C under an argon atmosphere. The morphology of layers is determined by reaction time. Understanding the factors that indicate the transition sequence and control the phase stability may provide insight into how the properties of titanium dioxide nanomaterials can be manipulated. A cross-sectional view of the samples reveals the complex arrangement of grains and their different shapes. HR-TEM and 3D Raman spectroscopy allowed to gain new insights into the structure formation. Observations of vibrational modes of anatase (101) and (001) surfaces allow quantitatively defining the percentage of facets exposition. Moreover, the spectra collected from different z-heights for the sample and the analysis of the integrated intensity ratio of Eg (144 cm−1) and A1g (514 cm−1) anatase bands show the grains' variable nature. The results present that the inner layer of the nanostructure is composed of more oriented crystallites, in contrast to the oxide/substrate interface and the sample surface. The model of the growth mechanism of a multilayered TiO2 scale formed on Ti has been proposed. Data availability statementThe raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Misinterpretation in microplastic detection in biological tissues: When 2D imaging is not enough

The presence of microplastics in the food chain is a public concern worldwide, and its analysis is an analytical challenge. In our research, we apply Raman imaging to study the presence of 1 μm polystyrene microplastics in cryosections of Mytilus galloprovincialis due to its wide geographic distribution, widespread occurrence in the food web, and general high presence in the environment. Ingested microplastics are accumulated in the digestive tract, but a large number can also be rapidly eliminated. Some authors state that the translocation of microplastics to the epithelial cells is possible, increasing the risk of microplastics transmission along the food chain. However, as seen in our study, a surface imaging approach (2D) is probably not enough to confirm the internalization of particles and avoid misinterpretation. In fact, while some microplastic particles were detected in the epithelium by 2D Raman imaging, further 3D Raman imaging analysis demonstrated that those particles were dragged from the lumens to the epithelium during sample preparation due to the blade drag effect of the cryotome, and subsequently located on the surface of the analyzed cryosection, discarding the translocation to the epithelial cells. This effect can also happen when the samples are fortuitously contaminated during sample preparation. Several research articles that use similar analytical techniques have shown the presence of microplastics in different types of tissue. It is not our intention to put such results in doubt, but the present work points out the necessity of appropriate three-dimensional analytical methods including data interpretation and the need to go a step further than just surface imaging analysis.

Sensing soluble molecules through SERS substrates in one-step procedure: unrevealing the Meiji woodblock printing materials

Ultrasensitive SERS substrates allowed us to detect complex mixtures of coloring components from Meiji Japanese woodblock prints (1868–1912). In museum settings, compositional analyses have limitations due to restrictions to sampling advised by conservators and curators for the adequate preservation of the objects. An additional layer of complexity is brought by the high heterogeneity of heritage materials, usually not resolved with commercial portable instruments. High-performance instruments for in situ analyses are seldom available in museums. Furthermore, the chambers of most instruments for morphological or chemical characterization accommodate small samples rather than large or medium-sized objects. The innovative sampling strategy herein proposed comprises the gentle touch-dry removal of small coloring molecules weakly bound to the surface of heritage objects, transferred through a silicone sampler to planar SERS substrates with selected solvents in a one-step procedure. The analytical protocol reduces the amount of sample necessary for reliable identification of color components down to nanograms. The selectivity of the solvents combined with the geometry of the planar SERS sensing devices produces reliable signals for molecular identification, with no need for incision or wetting of the printed material. Further, 3D Raman imaging allowed us to reach an unprecedented degree of molecular discrimination, advancing previously available minimally-invasive instrumental methods used in heritage science research. The validation with historical inks from Meiji woodblock prints led to the identification of soluble synthetic azo β-naphthols, barium sulfonic lakes, purple anilines, Prussian blue, glass arsenic sulfides and other traditional coloring media.

Temperature-mediated construction of a plantar pressure-relieving, thermally insulating, and biodegradable thick-walled cellulose sponge insole

Plantar pressure can be reduced and wearing comfort can be improved by using cushioning materials such as insoles. However, the biodegradation of most commercial insoles is difficult, incurring high disposal costs both economically and environmentally. Herein, we present a temperature-mediated approach for constructing a plantar pressure reducing, thermally insulating, and biodegradable thick-walled cellulose sponge (TWCS) insole from cotton linters. 3D Raman imaging and crystallographic test results demonstrated that freezing temperature affected the packing behavior of the dispersed cellulose at the ice crystal boundary, resulting in a marked distinction between wall thicknesses and crystallinity of sponges, from 0.36 μm and 0.39 to 7.74 μm and 0.46, at distinguishing temperatures (−25 ˚C, −78 ˚C, and − 198 ˚C). Benefiting from the thick-walled structure generated during the temperature-mediated interfacial assembly, the mechanical strength of the TWCS was significantly increased by 5.6 times to 569 kPa, accompanied by significantly enhanced structural stability, pressure-relieving, and thermal insulating characteristics. Subsequently, we designed TWCS as a biomass insole, and its performance was thoroughly examined using plantar pressure testing, thermal performance assessment, finite element simulation, and degradability investigations. The results demonstrate that TWCS can effectively decompress (1-fold pressure reduction), disperse external stresses, prevent heat loss and complete degradation within 50 days under biological conditions, and can potentially replace traditional petroleum-based sponge insoles.

Serial section Raman tomography with 10 times higher depth resolution than confocal Raman microscopy

AbstractConfocal Raman microscopy enables 3D imaging of various samples solely based on chemical contrast. However, optical artifacts impair resolution and image quality in subsurface imaging. With serial section Raman tomography, we show that serial ultrathin and semithin sectioning by ultramicrotomy can successfully be combined with subsequent confocal Raman imaging. This new 3D Raman imaging technique reaches a depth resolution of up to 100 nm, which is about 10‐fold better than in confocal Raman microscopy. Structurally complex and optically inhomogeneous samples can not only be imaged, but also be used to quantify structural parameters. Serial section Raman tomography is a promising method for materials science and possibly also for life sciences.

Microfluidic flow-through reactor and 3D Raman imaging for in situ assessment of mineral reactivity in porous and fractured porous media.

An in-depth understanding of dissolution and precipitation of minerals in porous and fractured porous media and the complex feedback on the transport of fluids is essential for various subsurface applications. In this context, we developed a novel non-destructive "lab-on-chip" approach for quantitative in situ assessments of mineralogical changes in porous media. Our experimental approach involves a microfluidic flow-through reactor of reactive homogeneous and heterogeneous (fractured) porous media coupled with high-resolution imaging. Here, the reactive medium consists of compacted celestine grains seeded in a reservoir within the microfluidic chip. This medium reacts with a barium chloride solution injected into the microreactor at a constant flow rate, leading to the dissolution of celestine and growth of barite. Various seeding processes of the mineral grains allow the creation of homogeneous reactive porous media or the introduction of large heterogeneities such as fractures. Hence, our approach enables high-resolution investigations of reactive transport in fractured porous media. The use of confocal Raman spectroscopic techniques enables the spatio-temporal visualization of the mineral transformation at the pore-scale in two- and three-dimensions. Moreover, advanced pore-scale modelling correlates the hydrological heterogeneities to the geochemical observations in the micro-reactor, which explains the observed discrepancies between homogeneous and heterogeneous reactive media. Eventually, the proposed methodology can be applied to other chemical systems to provide new insights into hydro-geochemical coupling in porous and fractured porous media as well as high-fidelity datasets to benchmark reactive transport codes that are currently under development.

In vivo uptake of gold nanoparticles by the diatom Stephanopyxis turris

Nanoparticle (NP) implementation in industry has increased during the last years. However, the consequences of NP release to the environment have not been fully understood yet and long-term effects are hardly predictable. Information regarding a possible NP uptake by organisms is urgently needed. Here, we investigate the uptake of presynthesized AuNPs of different diameters by living diatoms. To better understand the influence of NPs on diatoms, cells were monitored in vivo using Surface Enhanced Raman Spectroscopy (SERS). By 3D Raman imaging, AuNPs with diameters ≥50 nm could clearly be localized inside the cells, whereas smaller AuNPs were never detected in the cell interior. This indicates a size-dependent uptake mechanism that comes along with different toxicities. As diatoms present an essential source of the marine food chain, this observation is relevant not only for diatoms themselves but also for higher organisms.

Focus on lasers, imaging, and microscopy

Focus on lasers, imaging, and microscopy

Raman imaging as a new approach to identification of the mayenite group minerals

The mayenite group includes minerals with common formula Ca12Al14O32−x(OH)3x[W6−3x], where W = F, Cl, OH, H2O and x = 0–2. This distinction in the composition is associated with W site which may remain unoccupied or be occupied by negatively charged ions: OH−, F−, Cl−, as well as neutral molecules like H2O. However, there is no experimental approach to easily detect or differentiate mineral species within the mayenite group. Electron micro-beam facilities with energy- or wavelength-dispersive X-ray detectors, as most common tools in mineralogy, appear to be insufficient and do not provide a definite identification, especially, of hydroxylated or hydrated phases. Some solution provides typical Raman analysis ensuring identification of minerals and 3D Raman imaging as an innovative approach to distinguish various co-existing minerals of the mayenite group within a small area of the rock sample. Raman spectroscopy has also been successfully used for a determination of water type incorporated into the mineral structure as well as for a spatial distribution of phases by cluster approach analysis and/or integrated intensity analysis of bands in the hydroxyl region. In this study, Raman technique was for the first time used to reconstruct a 3D model of mayenite group mineral zonation, as well as to determine a way of water incorporation in the structure of these minerals. Moreover, for the first time, Raman data were correlated with alterations during the mineral-forming processes and used for reconstruction of the thermal history of studied rock. As a result, the influence of combustion gases has been proposed as a crucial factor responsible for the transformation between fluormayenite and fluorkyuygenite.

Challenges in application of Raman spectroscopy to biology and materials.

Raman spectroscopy has become an essential tool for chemists, physicists, biologists and materials scientists. In this article, we present the challenges in unravelling the molecule-specific Raman spectral signatures of different biomolecules like proteins, nucleic acids, lipids and carbohydrates based on the review of our work and the current trends in these areas. We also show how Raman spectroscopy can be used to probe the secondary and tertiary structural changes occurring during thermal denaturation of protein and lysozyme as well as more complex biological systems like bacteria. Complex biological systems like tissues, cells, blood serum etc. are also made up of such biomolecules. Using mice liver and blood serum, it is shown that different tissues yield their unique signature Raman spectra, owing to a difference in the relative composition of the biomolecules. Additionally, recent progress in Raman spectroscopy for diagnosing a multitude of diseases ranging from cancer to infection is also presented. The second part of this article focuses on applications of Raman spectroscopy to materials. As a first example, Raman spectroscopy of a melt cast explosives formulation was carried out to monitor the changes in the peaks which indicates the potential of this technique for remote process monitoring. The second example presents various modern methods of Raman spectroscopy such as spatially offset Raman spectroscopy (SORS), reflection, transmission and universal multiple angle Raman spectroscopy (UMARS) to study layered materials. Studies on chemicals/layered materials hidden in non-metallic containers using the above variants are presented. Using suitable examples, it is shown how a specific excitation or collection geometry can yield different information about the location of materials. Additionally, it is shown that UMARS imaging can also be used as an effective tool to obtain layer specific information of materials located at depths beyond a few centimeters.

Biological synthesis of gold nanoparticles by the diatom Stephanopyxis turris and in vivo SERS analyses

Cultivation of the sea water diatom Stephanopyxis turris (S. turris) in a gold salt containing medium leads to the formation of gold nanoparticles. These biosynthesized nanoparticles were characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). According to their diameter, they are classified into two groups with average diameters of around 30 and 10nm. It is demonstrated that both types of nanoparticles consist exclusively of gold. The location of the gold nanoparticles with respect to the diatom cell was revealed using surface enhanced Raman scattering (SERS). To ensure a reliable observation of SERS spectra in each measured cell, the concentration of the gold solution as well as the incubation time of S. turris in this solution were optimized. 3D Raman imaging of entire S. turris cells was performed to localize SERS spectra of compounds resulting from closely neighbored nanoparticles. Interestingly, intracellular SERS spectra were observed indicating the presence of gold nanoparticles inside S. turris cells. Further investigation shows that the SERS spectra generated by biosynthesized nanoparticles differ significantly from the conventional Raman spectrum of diatoms. While pigments are dominating the resonance enhanced Raman spectrum, SERS spectra exhibit a completely different appearance. Their bands coincide with bands in SERS spectra of various biologically relevant compounds, e.g., hydroxyurea and retinol. The biosynthesized gold nanoparticles therefore have a high potential for prospective in vivo research of algal metabolism and its regulation.

Cetuximab-conjugated nanodiamonds drug delivery system for enhanced targeting therapy and 3D Raman imaging.

In this study, a multicomponent nanodiamonds (NDs)-based targeting drug delivery system, cetuximab-NDs-cisplatin bioconjugate, combining both specific targeting and enhanced therapeutic efficacy capabilities, is developed and characterized. The specific targeting ability of cetuximab-NDs-cisplatin system on human liver hepatocellular carcinoma (HepG2) cells is evaluated through epidermal growth factor receptor (EGFR) blocking experiments, since EGFR is over-expressed on HepG2 cell membrane. Besides, cytotoxic evaluation confirms that cetuximab-NDs-cisplatin system could significantly inhibit the growth of HepG2 cells, and the therapeutic activity of this system is proven to be better than that of both nonspecific NDs-cisplatin conjugate and specific EGF-NDs-cisplatin conjugate. Furthermore, a 3-dimensional (3D) Raman imaging technique is utilized to visualize the targeting efficacy and enhanced internalization of cetuximab-NDs-cisplatin system in HepG2 cells, using the NDs existing in the bioconjugate as Raman probes, based on the characteristic Raman signal of NDs at 1332 cm-1 . These advantageous properties of cetuximab-NDs-cisplatin system propose a prospective imaging and treatment tool for further diagnostic and therapeutic purposes.

Forbidden mineral assemblage coesite‐disordered graphite in diamond‐bearing kyanite gneisses (Kokchetav Massif)

Confocal 2D and 3D Raman imaging was applied to SiO2 inclusions in kyanite porphyroblasts from diamond‐bearing kyanite gneisses of the Kokchetav Massif (Northern Kazakhstan). The study reveals a mineral assemblage of coesite‐disordered graphite which is forbidden in ultrahigh pressure‐high temperature (UHP‐HT) metamorphic complexes. Disordered graphite in coesite inclusions is optically undetectable and has been identified exclusively by Raman spectroscopy. Raman imaging of the forbidden coesite‐disordered graphite assemblage fails to detect bands assigned to fluid components (e.g. CO2, N2, CH4, H2Oliq), but the Raman spectra of fluid inclusions in kyanite, garnet and quartz demonstrate the presence of CO2, N2, CH4, H2Oliq and disordered graphite. The disordered graphite most likely formed by precipitation from a C―O―H fluid during the retrograde stage. The fluid was entrapped by kyanite with coesite inclusions near the peak metamorphic conditions. The lack of disordered graphite around diamond crystals in our samples does not support the generally accepted origin of disordered graphite in ultra high pressure metamorphic rocks by partial diamond graphitization during exhumation. Copyright © 2017 John Wiley & Sons, Ltd.

Unsaturated lipid bodies as a hallmark of inflammation studied by Raman 2D and 3D microscopy

Endothelial HMEC-1 cells incubated with pro-inflammatory cytokine TNF-α for 6 and 24 hours were studied as a model of inflammation using Raman imaging. Striking changes in distribution, composition and concentration of cellular lipids were observed after exposure to TNF-α compared to the control. In particular, 3D Raman imaging revealed a significant increase in the amount of lipid entities formed under inflammation. Lipid bodies were randomly distributed in the cytoplasm and two types of droplets were assembled: more saturated one, in spectral characteristics resembling phosphatidylcholine and saturated cholesteryl esters, observed also in the control, and highly unsaturated one, containing also cholesterols, being a hallmark of inflamed cells. The statistical analysis showed that the number of lipid bodies was significantly dependent on the exposure time to TNF-α. Overall, observed formation of unsaturated lipid droplets can be directly correlated with the increase in production of prostacyclins - endogenous inflammation mediators.

CVD growth and properties of boron phosphide on 3C-SiC

Improving the crystalline quality of boron phosphide (BP) is essential for realizing its full potential in semiconductor device applications. In this study, 3C-SiC was tested as a substrate for BP epitaxy. BP films were grown on 3C-SiC(100)/Si, 3C-SiC(111)/Si, and 3C-SiC(111)/4H-SiC(0001) substrates in a horizontal chemical vapor deposition (CVD) system. Films were produced with good crystalline orientation and morphological features in the temperature range of 1000–1200°C using a PH3+B2H6+H2 mixture. Rotational twinning was absent in the BP due to the crystal symmetry-matching with 3C-SiC. Confocal 3D Raman imaging of BP films revealed primarily uniform peak shift and peak widths across the scanned area, except at defects on the surface. Synchrotron white beam X-ray topography showed the epitaxial relationship between BP and 3C-SiC was (100)〈011〉BP||(100)〈011〉3C-SiC and (111)〈112̅〉BP||(111)〈112̅〉3C-SiC. Scanning electron microscopy, Raman spectroscopy and X-ray diffraction analysis indicated residual tensile strain in the films and improved crystalline quality at temperatures below 1200°C. These results indicated that BP properties could be further enhanced by employing high quality bulk 3C-SiC or 3C-SiC epilayers on 4H-SiC substrates.

3D Raman imaging of systemic endothelial dysfunction in the murine model of metastatic breast cancer.

It was recently reported in the murine model of metastatic breast cancer (4T1) that tumor progression and development of metastasis is associated with systemic endothelial dysfunction characterized by impaired nitric oxide (NO) production. Using Raman 3D confocal imaging with the analysis of the individual layers of the vascular wall combined with AFM endothelial surface imaging, we demonstrated that metastasis-induced systemic endothelial dysfunction resulted in distinct chemical changes in the endothelium of the aorta. These changes, manifested as a significant increase in the protein content (18 %) and a slight decrease in the lipid content (4 %), were limited to the endothelium and did not occur in the deeper layers of the vascular wall. The altered lipid to protein ratio in the endothelium, although more pronounced in the fixed vascular wall, was also observed in the freshly isolated unfixed vascular wall samples in the aqueous environment (12 and 7 % change of protein and lipid content, respectively). Our results support the finding that the metastasis induces systemic endothelial dysfunction that may contribute to cancer progression.Graphical Schematic illustration of methodology of sample preparation and measurementElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9436-9) contains supplementary material, which is available to authorized users.

Hierarchical 3D SERS Substrates Fabricated by Integrating Photolithographic Microstructures and Self‐Assembly of Silver Nanoparticles

Most of the surface-enhanced Raman scattering (SERS) substrates are 2D planar systems, which limits the SERS active area to a single Cartesian plane. Here, we fabricate 3D SERS substrates with the aim to break the traditional 2D SERS active area limitation, and to extend the SERS hotspots into the third dimension along the z-axis. Our 3D SERS substrates are tailored with increased SERS hotspots in the z-direction from tens of nanometers to tens of micrometers, increasing the hotspots in the z-direction by at least an order of magnitude larger than the confocal volume (~1 μm) of most Raman spectrometers. Various hierarchical 3D SERS-active microstructures are fabricated by combining 3D laser photolithography with Langmuir-Blodgett nanoparticle assembly. 3D laser photolithography creates microstructured platforms required to extend the SERS-active area into 3D, and the self-assembly of Ag nanoparticles ensures homogeneous coating of SERS-active Ag nanoparticles over the entire microstructured platforms. Large-area 3D Raman imaging demonstrates that homogeneous signals can be collected throughout the entire 3D SERS substrates. We vary the morphology, height, and inclination angles of the 3D microstructures, where the inclination angle is found to exhibit strong influence on the SERS signals. We also demonstrate a potential application of this hierarchical 3D SERS substrate in information tagging, storage and encryption as SERS micro-barcodes, where multiple micro-barcodes can be created within a single set of microstructures.