Typical spectral acquisition settings consisted of 1 s acquisitions with mW of power at the sample, depending on the prominence of auto-fluorescence background. Background contributions were removed in WiRE 4.
All spectra in the map were then averaged, creating a single representative spectrum per 2D Raman scan. DCLS was performed on these spectra using four reference spectra, one from each of the three targeted particles and the isotype control, resulting in a score for each Raman reporter for each averaged spectrum.
Each Raman reporter score was averaged from 12 imaged cells per condition, with the standard deviation representing the error. Bayesian statistical analysis was performed in the R statistical computing platform [ 28 ]. Prior to Bayesian fitting, cosmic ray removal was conducted on individual spectra from each Raman map.
All spectra in each map were then averaged, creating a single representative spectrum per 2D Raman scan. Bayesian linear regression was then performed using four reference spectra and a penalized cubic spline modeling the baseline as in Moores et al. For baseline modeling, baseline knots were placed 5 wavenumbers apart. For the prior distributions of the scores, truncated normal distributions with mean 0, standard deviation and truncation at 0 were selected as scores must be non-negative. Finally, for the variance of the errors, we chose an inverse-Gamma distribution with shape 0.
We obtained sequential Monte Carlo samples from the relevant posterior distribution for each spectrum [ 29 ]. Spectra were then grouped according to stimulation condition and 12 posterior sample members were averaged within each group to give average scores per reference spectra per stimulation condition.
Statistical analyses were performed using GraphPad Prism Version 6. Statistical significance was calculated using ordinary one-way ANOVA with post-hoc Tukey's test, and R 2 values were calculated using linear regression. We used Bayesian linear regression [ 30 ] to obtain posterior probabilities of differences between means from the average scores.
BFNP were designed and synthesized consisting of a surface-enhancing gold core, a Raman reporter to endow a unique Raman spectral signature, polyethylene glycol PEG for stability, biocompatibility and prolonged in vivo circulation times, and antibodies to provide target molecule specificity. The spectrum of each Raman reporter utilized is shown, with the unique spectral features used for their identification highlighted Figure S2 B. Darkfield images indicated the presence of BFNP on cells, appearing as small bright spots due to their unique scattering properties, whilst delineation of particle type was achievable via the Raman spectra collected.
Examples of individual spectra acquired from within the Raman maps are shown, confirming that the experimentally observed spectra from specific points in the Raman map match prototypical reference spectra for each of the three targeted probes Figure 1 D. Following stimulation, coronary artery endothelial cells CAEC express adhesion molecules detectable via immuno-SERS imaging in single and multiplex formats.
Optical images in B-C are darkfield images. Results are representative of 3 independent experiments. Information obtained from Raman imaging of the BFNP on fixed cells was used to develop a semi-quantitative approach to monitor changes in biomarker expressions. Human umbilical vein endothelial cells HUVEC were employed, which demonstrated a lower basal but similar induced expression of adhesion molecules in comparison with CAECs data not shown.
This single marker immunofluorescence quantification was as a standard with which to compare two multiplexed SERS quantification methodologies: one using direct classical least squares DCLS and the other using a Bayesian approach. Two quantification methods were explored as a standard method of quantification that is rigorous and robust enough for broad application; a unanimous model has yet to be defined due to the novelty of SERS quantification methodologies. Both quantification methodologies produced near-zero scores in all stimulation conditions explored for isotype control BFNP Figure S7.
The SERS detection approach was applied in human tissue ex vivo using a single Raman fiber probe-configured, custom-made spectroscopic system [ 27 ]. Fresh segments of human coronary arteries from patients undergoing heart transplantation were obtained for this study.
Vessels which did not present any evidence of atherosclerosis did not lead to the detection of significant adhesion molecules using any spectroscopic approach Figure S8. The artery shown in Figure 3 contained an atherosclerotic plaque, with tissue remodeling and lipid accumulation evident Figure 3 A , that also had an intact endothelium Figure 3 B. Adhesion molecule staining is shown in white; all cells were counterstained with Hoechst to identify nuclei blue. Cells were then subjected to SERS mapping. Optical images in B are darkfield images.
Each Raman reporter score was averaged from 12 imaged cells per condition. A single human coronary artery was isolated from the heart of a patient undergoing heart transplantation surgery. Sutures were then removed and the artery segment was thoroughly washed prior to SERS spectroscopy and subsequent analysis of morphology, expression of adhesion molecules and SERS mapping.
A Photos are shown of an intact upper left panel and dissected en face opened upper middle panel atherosclerotic artery. The plaque location is highlighted with a red circle. Following SERS spectroscopy analysis, the opened artery was bisected to separate atherosclerotic and non-atherosclerotic regions of the vessel; the cut location is highlighted with a black dashed line upper right panel.
Nuclei were counterstained using Hoechst blue.
D-E SERS spectroscopy was conducted on atherosclerotic red lines and non-atherosclerotic black lines regions of the intact D and opened E artery. The unique peaks and common peak used to identify each BFNP configuration are highlighted with a red arrow and dotted line, and labeled accordingly. Spectroscopy results are displayed as spectra averaged from at least 3 different points within the atherosclerotic and non-atherosclerotic regions.
Darkfield images are shown and the regions of non-atherosclerotic G and atherosclerotic H artery subject to SERS mapping are highlighted with yellow and red boxes corresponding to SERS maps on the right of each darkfield image. Optical images in G-H are darkfield images. To confirm the accuracy of these results, the artery was opened and the Raman spectroscopic investigation was repeated, allowing more rigorous control of the location being analyzed and improved sampling Figure 3 E.
Using the DCLS quantification strategy, the results obtained from this artery were quantified, producing a numerical trend towards increased adhesion molecule expression, but no change in isotype signal, when comparing atherosclerotic to non-atherosclerotic regions of tissue Figure 3 F. Furthermore, BFNP-related darkfield contrast at the endothelial surface of atherosclerotic tissue was visibly increased compared with that at non-atherosclerotic regions, suggesting far greater BFNP density at this tissue site.
In summary, these data confirm the suitability of this SERS-BFNP platform for simultaneous multiplexed detection of adhesion molecules in human atherosclerotic artery ex vivo. To achieve this, a humanized mouse model HA NSG model was used whereby NSG mice, which are amenable to xenografting due to their immunocompromised status [ 32 , 33 ], were engrafted with human subcutaneous adipose tissue. Adipose engraftment resulted in NSG mice containing viable and perfused human microvasculature Figure S9.
The presence of human vessels, which were identified microscopically in close proximity to the murine vasculature, was confirmed Figure 4 A. At 24 h post injection, there was little to no signal from BFNP in the blood, suggesting clearance from the circulation, with BFNP signal from the liver being similar for both isotype and targeted probes at 1 h and 24 h post-BFNP injection Figure S The peak common to all reporter molecules is present in all spectra from the targeted mice; in contrast, BFNP signals were not observed in mice receiving equivalent isotype controls.
Spectra obtained from 5 isotype and 5 targeted mice are shown Figure 4 D. Examples of individual SERS spectra acquired within microscopy images are shown, confirming that the experimentally observed spectra from specific points in the Raman image match prototypical reference spectra for each of the three targeted probes Figure 4 F.
In summary, these data demonstrate the successful application of a SERS-BFNP imaging approach in vivo for multiplexed non-invasive imaging of adhesion molecules. In this study, we have demonstrated the feasibility of a SERS-BFNP molecular imaging platform for non-invasive, simultaneous targeted multiplexed detection of adhesion molecules in vivo in the context of vascular inflammation. There is great interest in the ability to image specific inflammatory biomarkers from the clinical perspective, as inflammation plays fundamental roles in atherosclerosis, but at present there are significant limitations in the potential to observe these molecules in patients.
As such, this study will have important implications for potentiating the development of clinical SERS-BFNP molecular imaging systems in the cardiovascular setting and in any other pathology in need of multiplexed biomarker imaging. Molecule-specific BFNP were developed—a strategy employed in several other studies [ 16 , 17 , 19 - 22 , 35 ]—as a means of detecting adhesion molecules using SERS.
We have targeted adhesion molecules as a proof of concept, given that they are easily accessible to intravenously injected contrast agents. Supporting previous studies using BFNP as drug delivery systems [ 36 , 37 ], it was found that ICAMtargeted particles were internalized by CAECs, resulting in amplification of the SERS signal and presenting a mechanism that could be manipulated for theranostic drug delivery or improving imaging depth. The spectra shown are from 5 isotype vs. E In addition to immunofluorescence microscopy, excised adipose grafts were analyzed using SERS microscopy.
The colored circles in the Raman map E upper panel correlate to the acquired spectra shown in F above their respective reference spectra. The optical image in E is a darkfield image.
Quantification of adhesion molecule expression using the SERS-BFNP molecular imaging system was achieved and, in the process, demonstrated that both DCLS and Bayesian multiplexed quantification approaches correlated well with a classical immunofluorescence methodology in vitro. Building on the work of the Liu group [ 38 - 43 ], who have developed a semi-quantitative SERS-BFNP approach for tumor phenotyping, an isotype BFNP was included in the multiplex probe panel as an internal standard for non-specific binding.
Due to variable levels of non-specific binding observed in different tumor types, the phenotyping of tumors in the referenced studies required ratiometric normalization of biomarker scores to isotype BFNP scores. In comparison, normalization was not required in this study due to near zero scores for isotype-BFNP in all conditions analyzed. The ability to quantify vascular inflammatory biomarkers would be most advantageous, as it is the balance between pro- and anti-inflammatory mechanisms that will define pathological outcomes [ 2 , 44 , 45 ].
Raman spectroscopy imaging approaches, which focus on detecting the native spectral fingerprints of tissue components such as calcium and collagen, have demonstrated the ability to differentiate atherosclerotic and non-atherosclerotic arteries with reasonable success [ 46 - 48 ]. However, acquisition of Raman signals from atherosclerotic plaques in vivo requires the use of invasive intravascular probes due to the inherent lack of tissue penetration commonly associated with Raman spectroscopy.
By comparison, the use of targeted BFNP in a SERS approach greatly expands the number of biomarkers one could image, whilst providing the sensitivity needed to detect spectra through several centimeters of tissue, thus offering the potential for non-invasive use in superficial arteries [ 27 , 49 , 50 ]. To put the multiplexing potential into context, identification and discrimination of 10 non-targeted SERS nanoprobes has been demonstrated in vivo , making expansion of the number of target molecules simultaneously imaged a distinct possibility in the future [ 51 ].
Specifically, SERS spectroscopic detection of adhesion molecules was only achieved in atherosclerotic arteries, highlighting the potential for discriminating atherosclerotic from non-atherosclerotic tissue. The incorporation of additional biomarkers related to inflammation, angiogenesis or thrombotic pathways would allow increased sensitivity of detection and potentially plaque phenotyping in respect to detecting early stage lesions and atherosclerosis at high risk of causing a clinically significant event.
Furthermore, the DCLS quantification approach tested in vitro verified our interpretations of plaque vs. Published on Oct 20, SlideShare Explore Search You. Submit Search. Successfully reported this slideshow. We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads.
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Brand New: A new, unread, unused book in perfect condition with no missing or damaged pages. The First Book on CRS Microscopy Compared to conventional Raman microscopy, coherent Raman scattering CRS allows label-free imaging of living cells and tissues at video rate by enhancing the weak Raman signal through nonlinear excitation.
Series: Series in Cellular and Clinical Imaging Compared to conventional Raman microscopy, coherent Raman scattering (CRS) allows label-free imaging of. Read Coherent Raman Scattering Microscopy (Series in Cellular and Clinical Imaging) book reviews & author details and more at irelytuqypov.ml Free delivery on .
Edited by pioneers in the field and with contributions from a distinguished team of experts, Coherent Raman Scattering Microscopy explains how CRS can be used to obtain a point-by-point chemical map of live cells and tissues. In color throughout, the book starts by establishing the foundation of CRS microscopy. It discusses the principles of nonlinear optical spectroscopy, particularly coherent Raman spectroscopy, and presents the theories of contrast mechanisms pertinent to CRS microscopy.
The text then provides important technical aspects of CRS microscopy, including microscope construction, detection schemes, and data analyses. It concludes with a survey of applications that demonstrate how CRS microscopy has become a valuable tool in biomedicine. Due to its label-free, noninvasive examinations of living cells and organisms, CRS microscopy has opened up exciting prospects in biology and medicine from the mapping of 3D distributions of small drug molecules to identifying tumors in tissues.
An in-depth exploration of the theories, technology, and applications, this book shows how CRS microscopy has impacted human health and will deepen our understanding of life processes in the future. Day, Katrin F. Domke, Gianluca Rago, Erik M. Parekh, and Khaled A. Henry, R. Valle, M. Randolph, I. Kochevar, J. Winograd, Charles P. Buhman Index.