Advanced Microspatially Offset Raman Spectroscopy for Noninvasive Imaging of Concealed Texts and Figures Using Raman Signal, Fluorescence Emission, and Overall Spectral Intensity.

This study explores the possibility of using microspatially offset Raman spectroscopy (micro-SORS) imaging to reconstruct noninvasively letters and figures hidden by opaque layers. Micro-SORS experiments were conducted on mockup samples that mimic real situations encountered in the cultural heritage field, such as sealed letters with inaccessible text and original documents. Subsurface images were obtained using both the characteristic Raman bands of the hidden compounds and their different optical properties from the remaining matrix. In the latter case, contrast obtained through observing a difference in the overall spectral intensity and fluorescence profile rather than any specific Raman bands were used to track the images within the hidden layer. This approach opens new prospects for the use of micro-SORS in heritage science, with applications in the field that include the study of objects covered by opaque overlayers not only through their Raman signatures but also through differences in their optical properties (e.g., fluorescence emission, absorption).

Guided principal component analysis (GPCA): a simple method for improving detection of a known analyte.

There is increasing interest in the application of Raman spectroscopy in a medical setting, ranging from supporting real-time clinical decisions e.g. surgical margins to assisting pathologists with disease classification. However, there remain a number of barriers for adoption in the medical setting due to the increased complexity of probing highly heterogeneous, dynamic biological materials. This inherent challenge can also limit the deployment of higher level analytical approaches such as Artificial Intelligence (AI) including convolutional neural networks (CNN), as there is a lack of a ground truth required for training purposes i.e. in complex clinical samples. Principal component analysis (PCA) is an unsupervised data reduction approach (orthogonal linear transformation) that has been used extensively in spectroscopy for 30+ years, due to its capability to simplify analysis of complex spectroscopic data. However, due to PCA being unsupervised features will inherently appear mixed and their rank may vary between experiments. Here we propose Guided PCA (GPCA), a simple approach that allows PCA to be guided with spectral data to ensure a consistent rank of a key target moiety by the inclusion of a reference (guiding) spectrum to the data set. This simplifies analysis, increases robustness of PCA analysis and improves quantification and the limits of detection and decreases RMSE.

Non-destructive Monitoring of Dye Depth Profile in Mesoporous TiO2 Electrodes of Solar Cells with Micro-SORS.

The dye distribution within a photo-electrode is a key parameter in determining the performances of dye-sensitized photon-to-electron conversion devices, such as dye-sensitized solar cells (DSSCs). A traditional, depth profiling investigation by destructive means including cross-sectional sampling is unsuitable for large quality control applications in manufacturing processes. Therefore, a non-destructive monitoring of the dye depth profile is required, which is the first step toward a non-destructive evaluation of the internal degradation of the device in the field. Here, we present a conceptual demonstration of the ability to monitor the dye depth profile within the light active layer of DSSCs by non-destructive means with high chemical specificity using a recently developed non-destructive/non-invasive Raman method, micro-spatially offset Raman spectroscopy (micro-SORS). Micro-SORS is able to probe through turbid materials, providing the molecular identification of compounds located under the surface, without the need of resorting to a cross-sectional analysis. The study was performed on the photo-electrode of DSSCs. This represents the first demonstration of the micro-SORS concept in the solar cell area as well as, more generally, the application of micro-SORS to the thinnest layer to date. A sample set has been prepared with varying concentrations of the dye and the thickness of the matrix consisting of a titanium dioxide layer. The results showed that micro-SORS can unequivocally discriminate between the homogeneous and inhomogeneous dye depth profiles. Moreover, micro-SORS outcomes have been compared with the results obtained with destructive time-of-flight secondary ion mass spectrometry measurements. The results of the two techniques are in good agreement, confirming the reliability of micro-SORS analysis. Therefore, this study is expected to pave the way for establishing a wider and more effective monitoring capability in this important field.

Spatially offset Raman spectroscopy for biomedical applications.

In recent years, Raman spectroscopy has undergone major advancements in its ability to probe deeply through turbid media such as biological tissues. This progress has been facilitated by the advent of a range of specialist techniques based around spatially offset Raman spectroscopy (SORS) to enable non-invasive probing of living tissue through depths of up to 5 cm. This represents an improvement in depth penetration of up to two orders of magnitude compared to what can be achieved with conventional Raman methods. In combination with the inherently high molecular specificity of Raman spectroscopy, this has therefore opened up entirely new prospects for a range of new analytical applications across multiple fields including medical diagnosis and disease monitoring. This article discusses SORS and related variants of deep Raman spectroscopy such as transmission Raman spectroscopy (TRS), micro-SORS and surface enhanced spatially offset Raman spectroscopy (SESORS), and reviews the progress made in this field during the past 5 years including advances in non-invasive cancer diagnosis, monitoring of neurotransmitters, and assessment of bone disease.

Chemical Markers of Human Tendon Health Identified Using Raman Spectroscopy: Potential for In Vivo Assessment.

The purpose of this study is to determine whether age-related changes to tendon matrix molecules can be detected using Raman spectroscopy. Raman spectra were collected from human Achilles (n = 8) and tibialis anterior (n = 8) tendon tissue excised from young (17 ± 3 years) and old (72 ± 7 years) age groups. Normalised Raman spectra underwent principal component analysis (PCA), to objectively identify differences between age groups and tendon types. Certain Raman band intensities were correlated with levels of advanced glycation end-product (AGE) collagen crosslinks, quantified using conventional destructive biochemistry techniques. Achilles and tibialis anterior tendons in the old age group demonstrated significantly higher overall Raman intensities and fluorescence levels compared to young tendons. PCA was able to distinguish young and old age groups and different tendon types. Raman intensities differed significantly for several bands, including those previously associated with AGE crosslinks, where a significant positive correlation with biochemical measures was demonstrated. Differences in Raman spectra between old and young tendon tissue and correlation with AGE crosslinks provides the basis for quantifying age-related chemical modifications to tendon matrix molecules in intact tissue. Our results suggest that Raman spectroscopy may provide a powerful tool to assess tendon health and vitality in the future.

Spatially Offset Raman Spectroscopy-How Deep?

Spatially offset Raman spectroscopy (SORS) is a technique for interrogating the subsurface composition of turbid samples noninvasively. This study generically addresses a fundamental question relevant to a wide range of SORS studies, which is how deep SORS probes for any specific spatial offset when analyzing a turbid sample or, in turn, what magnitude of spatial offset one should select to probe a specific depth. This issue is addressed by using Monte Carlo simulations, under the assumption of negligible absorption, which establishes that the key parameter governing the extent of the probed zone for a point-like illumination and point-like collection SORS geometry is the reduced scattering coefficient of the medium. This can either be deduced from literature data or directly estimated from a SORS measurement by evaluating the Raman intensity profile from multiple spatial offsets. Once this is known, the extent of the probed zone can be determined for any specific SORS spatial offset using the Monte Carlo simulation results presented here. The proposed method was tested using experimental data on stratified samples by analyzing the signal detected from a thin layer that was moved through a stack of layers using both non-absorbing and absorbing samples. The proposed simple methodology provides important additional information on SORS measurements with direct relevance to a wide range of SORS applications including biomedical, pharmaceutical, security, forensics, and cultural heritage.

Estimating the Reduced Scattering Coefficient of Turbid Media Using Spatially Offset Raman Spectroscopy.

We propose a new method for estimating the reduced scattering coefficient, µs', of turbid homogeneous samples using Spatially Offset Raman Spectroscopy (SORS). The concept is based around the variation of Raman signal with SORS spatial offset that is strongly µs'-dependent, as such, permitting the determination of µs'. The evaluation is carried out under the assumptions that absorption is negligible at the laser and Raman wavelengths and µs' is approximately the same for those two wavelengths. These conditions are often satisfied for samples analyzed in the NIR region of the spectrum where SORS is traditionally deployed. Through a calibration procedure on a PTFE model sample, it was possible to estimate the µs' coefficient of different turbid samples with an error (RMSEP) below 18%. The knowledge of µs' in the NIR range is highly valuable for facilitating accurate numerical simulations to optimize illumination and collection geometries in SORS, to derive in-depth information about the properties of SORS measurements or in other photon applications, dependent on photon propagation in turbid media with general impact across fields such as biomedical, pharmaceutical, security, forensic, and cultural sciences.

Self-absorption corrected non-invasive transmission Raman spectroscopy (of biological tissue).

The first near infrared window in biological tissue (λ∼ 700-950 nm) is of great interest for its potential to safely deliver light based diagnosis and therapeutic interventions, especially in the burgeoning field of nano-theranostics. In this context, Raman spectroscopy is increasingly being used to provide rapid non-invasive chemical molecular analysis, including bulk tissue analysis by exploiting the near infrared window, with transmission Raman spectroscopy (TRS). The disadvantage of this approach, is that when probing depths of several centimetres self-attenuation artefacts are typically exhibited, whereby TRS spectra can suffer from relative changes in the "spectral features" due to differential absorption of Raman photons by the various constituents of biological tissues. Simply put, for a homogenous substance with increasing thickness, spectral variances occur due to the optical properties of the material and not through changes in the chemical environment. This can lead to misinterpretation of data, or features of interest become obscured due to the unwanted variance. Here we demonstrate a method to correct TRS data for this effect, which estimates the pathlengths derived from peak attenuation and uses expected optical properties to transform the data. In a validation experiment, the method reduced total Raman spectral intensity variances >5 fold, and improved specific peak ratio distortions 35×. This is an important development for TRS, Spatially Offset Raman Spectroscopy (SORS) and related techniques operating at depth in the near IR window; applicable to samples where there is large sample thickness and inter- and intra-sample thickness is variable i.e. clinical specimens from surgical procedures such as breast cancer. This solution is expected to yield lower detection limits and larger depths in future applications such as non-invasive breast cancer diagnosis in vivo.

Noninvasive Detection of Differential Water Content Inside Biological Samples Using Deep Raman Spectroscopy.

Here we conceptually demonstrate the capability of deep Raman spectroscopy to noninvasively monitor changes in the water content within biological tissues. Water was added by injection into an isolated tissue volume (a 20 mm diameter disk of 5 mm thickness) representing a 20% increase in the overall mass, which was equivalent to a 5% increase in the water/tissue content. The elevated water content was detected through a larger volume of tissue with a total thickness of approximately 12 mm and a spiked tissue segment located in its center using transmission Raman spectroscopy (TRS) by monitoring the change of the OH (∼3390 cm-1) Raman band area (3350-3550 cm-1 spectral region) after being normalized to the neighboring CH stretching band. The tissue sample was raster scanned with TRS to yield a spatial map of the water concentration within the sample encompassing the spiked tissue zone. The mapping revealed the presence and location of the spiked region. The results provide the first conceptual demonstration using a deep Raman-based architecture, which can be used noninvasively for the detection of an elevated water content deep within biological tissues. It is envisaged that this concept could play a role in rapid in vivo detection and localization of cancerous lesions (generally exhibiting a higher water content) beneath the tissue surface.

Plasmonic Nanoassemblies: Tentacles Beat Satellites for Boosting Broadband NIR Plasmon Coupling Providing a Novel Candidate for SERS and Photothermal Therapy.

Optical theranostic applications demand near-infrared (NIR) localized surface plasmon resonance (LSPR) and maximized electric field at nanosurfaces and nanojunctions, aiding diagnosis via Raman or optoacoustic imaging, and photothermal-based therapies. To this end, multiple permutations and combinations of plasmonic nanostructures and molecular "glues" or linkers are employed to obtain nanoassemblies, such as nanobranches and core-satellite morphologies. An advanced nanoassembly morphology comprising multiple linear tentacles anchored onto a spherical core is reported here. Importantly, this core-multi-tentacle-nanoassembly (CMT) benefits from numerous plasmonic interactions between multiple 5 nm gold nanoparticles (NPs) forming each tentacle as well as tentacle to core (15 nm) coupling. This results in an intense LSPR across the "biological optical window" of 650-1100 nm. It is shown that the combined interactions are responsible for the broadband LSPR and the intense electric field, otherwise not achievable with core-satellite morphologies. Further the sub 80 nm CMTs boosted NIR-surface-enhanced Raman scattering (SERS), with detection of SERS labels at 47 × 10-9 m, as well as lower toxicity to noncancerous cell lines (human fibroblast Wi38) than observed for cancerous cell lines (human breast cancer MCF7), presents itself as an attractive candidate for use as biomedical theranostics agents.

Non-invasive depth determination of inclusion in biological tissues using spatially offset Raman spectroscopy with external calibration.

Spatially offset Raman spectroscopy (SORS) allows chemical characterisation of biological tissues at depths of up to two orders of magnitude greater than conventional Raman spectroscopy. In this study, we demonstrate the use of SORS for the non-invasive prediction of depth of an inclusion within turbid media (e.g. biological tissues) using only external calibration data sets, thus extending our previous approach that required internal calibration. As with the previous methodology, the concept is based on relative changes in Raman band intensities of the inclusion that are directly related to the path length of Raman photons travelling through the medium thereby encoding the information of depth of the inclusion. However, here the calibration model is created using data only from external measurements performed at the tissue surface. This new approach facilitates a fully non-invasive methodology applicable potentially to in vivo medical diagnosis without any a priori knowledge. Monte Carlo simulations of photon propagation have been used to provide insight into the relationship between the spatial offset and the photon path lengths inside the tissues enabling one to derive a general scaling factor permitting the use of spatial offset measurements for the depth prediction. The approach was validated by predicting the depth of surface-enhanced Raman scattering (SERS) labelled nanoparticles (NPs) acting as inclusions inside a slab of ex vivo porcine tissue yielding an average root mean square error of prediction of 7.3% with respect to the overall tissue thickness. Our results pave the way for future non-invasive deep Raman spectroscopy in vivo by enabling, for example, the localisation of cancer lesions or cancer biomarkers in early disease diagnosis and targeted treatments.

Detection of Age-Related Changes in Tendon Molecular Composition by Raman Spectroscopy-Potential for Rapid, Non-Invasive Assessment of Susceptibility to Injury.

The lack of clinical detection tools at the molecular level hinders our progression in preventing age-related tendon pathologies. Raman spectroscopy can rapidly and non-invasively detect tissue molecular compositions and has great potential for in vivo applications. In biological tissues, a highly fluorescent background masks the Raman spectral features and is usually removed during data processing, but including this background could help age differentiation since fluorescence level in tendons increases with age. Therefore, we conducted a stepwise analysis of fluorescence and Raman combined spectra for better understanding of the chemical differences between young and old tendons. Spectra were collected from random locations of vacuum-dried young and old equine tendon samples (superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT), total n = 15) under identical instrumental settings. The fluorescence-Raman spectra showed an increase in old tendons as expected. Normalising the fluorescence-Raman spectra further indicated a potential change in intra-tendinous fluorophores as tendon ages. After fluorescence removal, the pure Raman spectra demonstrated between-group differences in CH2 bending (1450 cm-1) and various ring-structure and carbohydrate-associated bands (1000-1100 cm-1), possibly relating to a decline in cellular numbers and an accumulation of advanced glycation end products in old tendons. These results demonstrated that Raman spectroscopy can successfully detect age-related tendon molecular differences.

Subsurface Chemically Specific Measurement of pH Levels in Biological Tissues Using Combined Surface-Enhanced and Deep Raman.

There is much interest in using nanosensors to monitor biologically relevant species such as glucose, or cellular pH, as these often become dysregulated in diseases such as cancer. This information is often inaccessible at depth in biological tissue, due to the highly scattering nature of tissue. Here we show that gold nanoparticles labeled with pH-sensitive reporter molecules can monitor pH at depth in biological tissues. This was achieved using deep Raman spectroscopy (spatially offset Raman and transmission Raman) in combination with surface-enhanced Raman spectroscopy, allowing chemical information to be retrieved significantly deeper than conventional Raman spectroscopy permits. Combining these approaches with chemometrics enabled pH changes to be monitored with an error of ±âˆ¼0.1 pH units noninvasively through 22 mm of soft tissue. This development opens the opportunity for the next generation of light-based medical diagnostic methods, such as monitoring of cancers, known to significantly alter pH levels.

Spatially Offset and Transmission Raman Spectroscopy for Determination of Depth of Inclusion in Turbid Matrix.

We propose an approach for the prediction of the depth of a single buried object within a turbid medium combining spatially offset Raman spectroscopy (SORS) and transmission Raman spectroscopy (TRS) and relying on differential attenuation of individual Raman bands brought about by the spectral variation of matrix absorption (and scattering). The relative degree of the Raman band changes is directly related to the path length of Raman photons traveling through the medium, thereby encoding the information on the depth of the object within the matrix. Through a calibration procedure with root mean square error of calibration (RMSEC) = 3.4%, it was possible to predict the depth of a paracetamol (acetaminophen) inclusion within a turbid matrix consisting of polyethylene (PE) by monitoring the relative intensity of two Raman bands of paracetamol exhibiting differential absorption by the matrix. The approach was shown to be largely insensitive to variations of the amount of the inclusion (paracetamol) and to the overall thickness of the turbid matrix (PE) with a root mean square error of prediction (RMSEP) maintained below 10% for the tested cases. This represents a major advantage over previously demonstrated comparable depth determination Raman approaches (with the exception of full Raman tomography requiring complex mathematical reconstruction algorithms). The obtained experimental data validate the proposed approach as an effective tool for the noninvasive determination of the depth of buried objects in turbid media with potential applications including determining noninvasively the depth of a lesion in cancer diagnosis in vivo.

Direct monitoring of light mediated hyperthermia induced within mammalian tissues using surface enhanced spatially offset Raman spectroscopy (T-SESORS).

Here we demonstrate light mediated heating of nanoparticles confined deep inside mammalian tissue, whilst directly monitoring their temperature non-invasively using a form of deep Raman spectroscopy, T-SESORS. One of the main barriers to the introduction of photo-thermal therapies (PTT) has been recognised as the inability to directly monitor the local temperature deep within the tissue at the point of therapy. Here Au nanoparticles with a Raman reporter molecule (temperature reporters) are used in combination with Au nanoshells (heat mediators) to provide simultaneously heating under NIR illumination and direct spectroscopic monitoring of local temperature deep within mammalian tissues. The surface enhanced Raman signal was read out at the tissue surface using a transmission geometry in this example and the temperature of the tissue was ascertained from the anti-Stokes to Stokes Raman reporter. This approach opens the prospect of non-invasive hyperthermia treatments with direct temperature feedback from deep inside within tissue, where nanoparticles can be used to both provide localised heating and accurately monitor the local temperature.

Fast Raman spectral mapping of highly fluorescing samples by time-gated spectral multiplexed detection.

We present a time-gated Raman micro-spectroscopy technique suitable for fast Raman mapping of samples eliciting large laser-induced fluorescence backgrounds. To achieve the required time resolution for effective fluorescence rejection, a picosecond pulsed laser and a single-photon avalanche diode were used. A module consisting of a spectrometer, digital micromirror device, and two prisms was used for high-resolution spectral filtering and multiplexing, which is required for a high chemical specificity and short integration times. With this instrument, we demonstrated time-gated Raman imaging of highly fluorescent samples, achieving acquisition times as short as 3 min for 40×40 pixel resolution images.

Noninvasive Determination of Depth in Transmission Raman Spectroscopy in Turbid Media Based on Sample Differential Transmittance.

Here we propose a simple noninvasive approach to determine the depth of a buried object using transmission Raman spectroscopy. In accordance with theory, the photons arising from spectral peaks that are suitably separated will be subjected to different optical properties in the media through which they travel. These differences can impact the relative intensities of Raman peaks as a function of the transmission path length, thereby the depth of signal generation is inherently encoded in the spectra. In a proof-of-concept study, through only external calibrations, it was possible to accurately predict the depth of Polytetrafluoroethylene (PTFE) layer purely on the basis of relative intensity of two peaks in a predominantly absorbing solution Indian ink (0.1 µL/mL; RMSE 0.42 mm) and a scattering solution (RMSE 0.50 mm). This simple approach offers the possibility to noninvasively identify the depth of a buried object, such as breast calcifications, using simple transmission measurement geometries for the first time.

Investigation of Heterogeneous Painted Systems by Micro-Spatially Offset Raman Spectroscopy.

A recently developed technique of Micro-Spatially Offset Raman Spectroscopy (micro-SORS) extends the applicability of Raman spectroscopy to probing thin, highly diffusely scattering layers such as stratified paint samples, enabling their nondestructive chemical characterization. The technique has a wide applicability across areas such as cultural heritage, polymer research, forensics, and biological fields; however, currently, it suffers from a major unaddressed issue related to its ineffectiveness with highly heterogeneous samples. In this paper, we address this unmet need while demonstrating an effective strategy to probe such samples, involving a mapping on scales substantially larger than the scale of heterogeneity. This approach provides an effective means of obtaining robust and representative micro-SORS datasets from which sample composition can be effectively deduced, even in these extreme scenarios. The approach is compared with a basic point collection approach on two-layer paint systems where different layers-top, bottom, or both-are heterogeneous. The study has particular relevance to cultural heritage, where heterogeneous layers are often encountered with painted stratigraphies.

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.

Development of a Fiber-Optics Microspatially Offset Raman Spectroscopy Sensor for Probing Layered Materials.

Microspatially offset Raman spectroscopy (micro-SORS) has been proposed as a valuable approach to sample molecular information from layers that are covered by a turbid (nontransparent) layer. However, when large magnifications are involved, the approach is not straightforward, as spatial constraints exist to position the laser beam and the objective lens with the external beam delivery or, with internal beam delivery, the maximum spatial offset achievable is restricted. To overcome these limitations, we propose here a prototype of a new micro-SORS sensor, which uses bare glass fibers to transfer the laser radiation to the sample and to collect the Raman signal from a spatially offset zone to the Raman spectrometer. The concept also renders itself amenable to remote delivery and to the miniaturization of the probe head which could be beneficial for special applications, e.g., where access to sample areas is restricted. The basic applicability of this approach was demonstrated by studying several layered structure systems. Apart from proving the feasibility of the technique, also, practical aspects of the use of the prototype sensor are discussed.