Distorted Phthalocyanines by Click Chemistry: Photoacoustic, Photothermal, and Surface-Enhanced Resonance Raman Studies.

Distortion of nominally planar phthalocyanine macrocycles affects the excited state dynamics in that most of the excited-state energy decays through internal conversion. A click-type annulation reaction on a perfluorophthalocyanine platform appending a seven-membered ring to the ß-positions on one or more of the isoindoles distorts the macrocycle and modulates solubility. The distorted derivative enables photoacoustic imaging, photothermal effects, and strong surface-enhanced resonance Raman signals.

Detection of low concentrations of ampicillin in milk.

Ampicillin, a common antibiotic, is detected at trace concentrations in milk using surface enhanced Raman spectroscopy in a microfluidic device, using less than 20 µL of sample, in 10 minutes, with minimal off-chip preparation. The device is configured so as to favor the interaction of the analyte with colloidal silver, and the optimization of the aggregation of the silver nanoparticles so as to increase the SERS intensity and the consequential sensitivity of analyte detection.

Discrete free-surface millifluidics for rapid capture and analysis of airborne molecules using surface-enhanced Raman spectroscopy.

A lithography-free, low-cost, free-surface millifluidic device is reported using discrete liquid interfaces for capturing and detecting gas-phase analyte molecules at low partial pressures out of a gas flow of time-varying composition. The architecture, based on segmented flow, consists of alternating regions of liquid and gas wherein the liquid regions contain surface-enhanced Raman spectroscopy (SERS)-active silver nanoparticles, while the gas regions contain trace quantities of vapor-phase analyte, thereby controlling and optimizing transport and mixing of the gas-phase analyte with the liquid phase. Once absorbed in the liquid phase, the entrained analyte molecules induce aggregation of the aqueous silver nanoparticles. The resulting aggregates consisting of nanoparticles and adsorbed analyte molecules produce intense SERS spectra that reliably identify the absorbed analyte in real time. The approach can be used to determine the time-variable trace chemical composition of a gas stream with applications in, for example, environmental monitoring and online industrial process monitoring, or as a SERS-based detector following gas chromatographic separation. The operation of the system is demonstrated using 4-aminobenzenethiol vapor at 750 ppb, and the detection response time is <2 min.

Assessment of volatile emissions by aging books.

Indoor air quality (IAQ) has attracted a lot of attention due to its complexity and direct effect on human health. Indoor settings in libraries entail various volatile organic compounds (VOCs) linked to the aging and degradation of print material. The effect of the storage environment on paper life expectancy was investigated by targeting the VOC emissions of old and new books using headspace solid phase micro extraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) analysis. "Sniffing" of book degradation markers showed both ubiquitously and infrequently occurring VOCs. Old book "degradomics" revealed mostly alcohols (57%) and ethers (12%), whereas new books resulted mainly to ketones (40%) and aldehydes (21%). Chemometric processing of the results with principal component analysis (PCA) corroborated our initial observations and was able to discriminate the books by age into three groups: very old books (from the 1600 s to mid-1700), old books (from the 1800s to the early 1900s), and modern books (from the mid-twentieth century onwards) based on their gaseous markers. The measured mean concentrations of selected VOCs (acetic acid, furfural, benzene, and toluene) were below the respective guidelines set for similar places (i.e. museums). The applied non-invasive, green analytical methodology (HS-SPME-GC/MS) can assist librarians, stakeholders, and researchers to evaluate the IAQ, as well as the degree of degradation, and take the appropriate measures for book restoration and monitoring protocols.

Aggregation kinetics of SERS-active nanoparticles in thermally stirred sessile droplets.

The aggregation kinetics of silver nanoparticles in sessile droplets were investigated both experimentally and through numerical simulations as a function of temperature gradient and evaporation rate, in order to determine the hydrodynamic and aggregation parameters that lead to optimal surface-enhanced Raman spectroscopic (SERS) detection. Thermal gradients promote effective stirring within the droplet. The aggregation reaction ceases when the solvent evaporates forming a circular stain consisting of a high concentration of silver nanoparticle aggregates, which can be interrogated by SERS leading to analyte detection and identification. We introduce the aggregation parameter, Γa ≡ τ(evap)/τ(a), which is the ratio of the evaporation to the aggregation time scales. For a well-stirred droplet, the optimal condition for SERS detection was found to be Γ(a,opt) = kc(NP)τ(evap) ≈ 0.3, which is a product of the dimerization rate constant (k), the concentration of nanoparticles (cNP), and the droplet evaporation time (τ(evap)). Near maximal signal (over 50% of maximum value) is observed over a wide range of aggregation parameters 0.05 < Γa < 1.25, which also defines the time window during which trace analytes can be easily measured. The results of the simulation were in very good agreement with experimentally acquired SERS spectra using gas-phase 1,4-benzenedithiol as a model analyte.

Organ-on-chip systems as a model for nanomedicine.

Nanomedicine is giving rise to increasing numbers of successful drugs, including cancer treatments, molecular imaging agents, and novel vaccine formulations. However, traditionally available model systems offer limited clinical translation and, compared to the number of preclinical studies, the approval rate of nanoparticles (NPs) for clinical use remains disappointingly low. A new paradigm of modeling biological systems on microfluidic chips has emerged in the last decade and is being gradually adopted by the nanomedicine community. These systems mimic tissues, organs, and diseases like cancer, on devices with small physical footprints and complex geometries. In this review, we report studies that used organ-on-chip approaches to study the interactions of NPs with biological systems. We present examples of NP toxicity studies, studies using biological NPs such as viruses, as well as modeling biological barriers and cancer on chip. Organ-on-chip systems present an exciting opportunity and can provide a renewed direction for the nanomedicine community.

Probing Aberrantly Glycosylated Mucin 1 in Breast Cancer Extracellular Vesicles.

Aberrantly glycosylated mucin 1 is a critical prognostic biomarker in breast epithelial cancers. Hypoglycosylated mucin 1 coats the surface of the cancer cells, where O-glycans are predominantly linked via an N-acetylgalactosamine moiety (GalNAc). Cancer cell-derived extracellular vesicles (EVs) carry biomarkers from parent cancer cells to the recipient cells and, therefore, could potentially be leveraged for diagnostics and noninvasive disease monitoring. We devised a label-free approach for identifying glycoprotein mucin 1 overexpression on breast cancer EVs. While exploring a plethora of biochemical (enzyme-linked immunosorbent assay, flow cytometry, and SDS-PAGE) and label-free biophysical techniques (circular dichroism and infrared spectroscopy (IR)) along with multivariate analysis, we discovered that mucin 1 is significantly overexpressed in breast cancer EVs and aberrant glycosylation in mucin 1 could be critically addressed using IR and multivariate analysis targeting the GalNAc sugar. This approach emerges as a convenient and comprehensive method of distinguishing cancer EVs from normal samples and holds potential for nonintrusive breast cancer liquid biopsy screening.

Correction: Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Correction for 'Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation' by Chrysafis Andreou et al., Nanoscale Horiz., 2022, 7, 1540-1552, https://doi.org/10.1039/d2nh00331g.

Near-Infrared Phototheranostic Iron Pyrite Nanocrystals Simultaneously Induce Dual Cell Death Pathways via Enhanced Fenton Reactions in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is considered more aggressive with a poorer prognosis than other breast cancer subtypes. Through systemic bioinformatic analyses, we established the ferroptosis potential index (FPI) based on the expression profile of ferroptosis regulatory genes and found that TNBC has a higher FPI than non-TNBC in human BC cell lines and tumor tissues. To exploit this finding for potential patient stratification, we developed biologically amenable phototheranostic iron pyrite FeS2 nanocrystals (NCs) that efficiently harness near-infrared (NIR) light, as in photovoltaics, for multispectral optoacoustic tomography (MSOT) and photothermal ablation with a high photothermal conversion efficiency (PCE) of 63.1%. Upon NIR irradiation that thermodynamically enhances Fenton reactions, dual death pathways of apoptosis and ferroptosis are simultaneously triggered in TNBC cells, comprehensively limiting primary and metastatic TNBC by regulating p53, FoxO, and HIF-1 signaling pathways and attenuating a series of metabolic processes, including glutathione and amino acids. As a unitary phototheranostic agent with a safe toxicological profile, the nanocrystal represents an effective way to circumvent the lack of therapeutic targets and the propensity of multisite metastatic progression in TNBC in a streamlined workflow of cancer management with an integrated image-guided intervention.

Multiplexed imaging in oncology.

In oncology, technologies for clinical molecular imaging are used to diagnose patients, establish the efficacy of treatments and monitor the recurrence of disease. Multiplexed methods increase the number of disease-specific biomarkers that can be detected simultaneously, such as the overexpression of oncogenic proteins, aberrant metabolite uptake and anomalous blood perfusion. The quantitative localization of each biomarker could considerably increase the specificity and the accuracy of technologies for clinical molecular imaging to facilitate granular diagnoses, patient stratification and earlier assessments of the responses to administered therapeutics. In this Review, we discuss established techniques for multiplexed imaging and the most promising emerging multiplexing technologies applied to the imaging of isolated tissues and cells and to non-invasive whole-body imaging. We also highlight advances in radiology that have been made possible by multiplexed imaging.

Label-Free Sensing with Metal Nanostructure-Based Surface-Enhanced Raman Spectroscopy for Cancer Diagnosis.

Surface-Enhanced Raman Spectroscopy (SERS) is a powerful analytical technique for the detection of small analytes with great potential for medical diagnostic applications. Its high sensitivity and excellent molecular specificity, which stems from the unique fingerprint of molecular species, have been applied toward the detection of different types of cancer. The noninvasive and rapid detection offered by SERS highlights its applicability for point-of-care (PoC) deployment for cancer diagnosis, screening, and staging, as well as for predicting tumor recurrence and treatment monitoring. This review provides an overview of the progress in label-free (direct) SERS-based chemical detection for cancer diagnosis with the main focus on the advances in the design and preparation of SERS substrates on the basis of metal nanoparticle structures formed via bottom-up strategies. It begins by introducing a synopsis of the working principles of SERS, including key chemometric approaches for spectroscopic data analysis. Then it introduces the advances of label-free sensing with SERS in cancer diagnosis using biofluids (blood, urine, saliva, sweat) and breath as the detection media. In the end, an outlook of the advances and challenges in cancer diagnosis via SERS is provided.

Advances in Surface Enhanced Raman Spectroscopy for in Vivo Imaging in Oncology.

In the last two decades, the application of surface enhanced Raman scattering (SERS) nanoparticles for preclinical cancer imaging has attracted increasing attention. Raman imaging with SERS nanoparticles offers unparalleled sensitivity, providing a platform for molecular targeting, and granting multiplexed and multimodal imaging capabilities. Recent progress has been facilitated not only by the optimization of the SERS contrast agents themselves, but also by the developments in Raman imaging approaches and instrumentation. In this article, we review the principles of Raman scattering and SERS, present advances in Raman instrumentation specific to cancer imaging, and discuss the biological means of ensuring selective in vivo uptake of SERS contrast agents for targeted, multiplexed, and multimodal imaging applications. We offer our perspective on areas that must be addressed in order to facilitate the clinical translation of SERS contrast agents for in vivo imaging in oncology.

Visualizing surface marker expression and intratumoral heterogeneity with SERRS-NPs imaging.

Cell surface marker expression in tumors dictates the selection of therapeutics, therapy response, and survival. However, biopsies are invasive, sample only a small area of the tumor landscape and may miss significant areas of heterogeneous expression. Here, we investigated the potential of antibody-conjugated surface-enhanced resonance Raman scattering nanoparticles (SERRS-NPs) to depict and quantify high and low tumoral surface marker expression, focusing on the surface markers epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in an intracerebral and peripheral setting with an inter- and intratumoral comparison of Raman signal intensities. Methods: ICR-Prkdc mice were injected with glioblastoma, epidermoid carcinoma, or breast tumor cell lines intracerebrally and peripherally. SERRS-NPs were functionalized with cetuximab or trastuzumab and administered via tail vein injection. Raman imaging was performed 18 hours post-injection in excised tumors and in vivo through the skull. Tumors were then fixed and processed for immunohistochemical evaluation. Results: Confirmed by MRI and immunohistochemistry for EGFR and HER2, our results demonstrate that antibody-conjugated SERRS-NPs go beyond the delineation of a tumor and offer clear and distinct Raman spectra that reflect the distribution of the targeted surface marker. The intensity of the SERRS-NP signal accurately discriminated high- versus low-expressing surface markers between tumors, and between different areas within tumors. Conclusion: Biopsies can be highly invasive procedures and provide a limited sample of molecular expression within a tumor. Our nanoparticle-based Raman imaging approach offers the potential to provide non-invasive and more comprehensive molecular imaging and an alternative to the current clinical gold standard of immunohistochemistry.

Colon Cancer: From Epidemiology to Prevention.

Colorectal cancer (CRC) is one of the most prevalent cancers affecting humans, with a complex genetic and environmental aetiology. Unlike cancers with known environmental, heritable, or sex-linked causes, sporadic CRC is hard to foresee and has no molecular biomarkers of risk in clinical use. One in twenty CRC cases presents with an established heritable component. The remaining cases are sporadic and associated with partially obscure genetic, epigenetic, regenerative, microbiological, dietary, and lifestyle factors. To tackle this complexity, we should improve the practice of colonoscopy, which is recommended uniformly beyond a certain age, to include an assessment of biomarkers indicative of individual CRC risk. Ideally, such biomarkers will be causal to the disease and potentially modifiable upon dietary or therapeutic interventions. Multi-omics analysis, including transcriptional, epigenetic as well as metagenomic, and metabolomic profiles, are urgently required to provide data for risk analyses. The aim of this article is to provide a perspective on the multifactorial derailment of homeostasis leading to the initiation of CRC, which may be explored via multi-omics and Gut-on-Chip analysis to identify much-needed predictive biomarkers.

Multiplexed molecular imaging with surface enhanced resonance Raman scattering nanoprobes reveals immunotherapy response in mice via multichannel image segmentation.

Visualizing the presence and distribution of multiple specific molecular markers within a tumor can reveal the composition of its microenvironment, inform diagnosis, stratify patients, and guide treatment. Raman imaging with multiple molecularly-targeted surface enhanced Raman scattering (SERS) nanoprobes could help investigate emerging cancer treatments preclinically or enable personalized treatment assessment. Here, we report a comprehensive strategy for multiplexed imaging using SERS nanoprobes and machine learning (ML) to monitor the early effects of immune checkpoint blockade (ICB) in tumor-bearing mice. We used antibody-functionalized SERS nanoprobes to visualize 7 + 1 immunotherapy-related targets simultaneously. The multiplexed images were spectrally resolved and then spatially segmented into superpixels based on the unmixed signals. The superpixels were used to train ML models, leading to the successful classification of mice into treated and untreated groups, and identifying tumor regions with variable responses to treatment. This method may help predict treatment efficacy in tumors and identify areas of tumor variability and therapy resistance.

Resveratrol loaded polymeric micelles for theranostic targeting of breast cancer cells.

Treatment of breast cancer underwent extensive progress in recent years with molecularly targeted therapies. However, non-specific pharmaceutical approaches (chemotherapy) persist, inducing severe side-effects. Phytochemicals provide a promising alternative for breast cancer prevention and treatment. Specifically, resveratrol (res) is a plant-derived polyphenolic phytoalexin with potent biological activity but displays poor water solubility, limiting its clinical use. Here we have developed a strategy for delivering res using a newly synthesized nano-carrier with the potential for both diagnosis and treatment. Methods: Res-loaded nanoparticles were synthesized by the emulsion method using Pluronic F127 block copolymer and Vitamin E-TPGS. Nanoparticle characterization was performed by SEM and tunable resistive pulse sensing. Encapsulation Efficiency (EE%) and Drug Loading (DL%) content were determined by analysis of the supernatant during synthesis. Nanoparticle uptake kinetics in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in MCF-10A breast epithelial cells were evaluated by flow cytometry and the effects of res on cell viability via MTT assay. Results: Res-loaded nanoparticles with spherical shape and a dominant size of 179±22 nm were produced. Res was loaded with high EE of 73±0.9% and DL content of 6.2±0.1%. Flow cytometry revealed higher uptake efficiency in breast cancer cells compared to the control. An MTT assay showed that res-loaded nanoparticles reduced the viability of breast cancer cells with no effect on the control cells. Conclusions: These results demonstrate that the newly synthesized nanoparticle is a good model for the encapsulation of hydrophobic drugs. Additionally, the nanoparticle delivers a natural compound and is highly effective and selective against breast cancer cells rendering this type of nanoparticle an excellent candidate for diagnosis and therapy of difficult to treat mammary malignancies.

Structurally symmetric near-infrared fluorophore IRDye78-protein complex enables multimodal cancer imaging.

Rationale: Most contemporary cancer therapeutic paradigms involve initial imaging as a treatment roadmap, followed by the active engagement of surgical operations. Current approved intraoperative contrast agents exemplified by indocyanine green (ICG) have a few drawbacks including the inability of pre-surgical localization. Alternative near-infrared (NIR) dyes including IRDye800cw are being explored in advanced clinical trials but often encounter low chemical yields and complex purifications owing to the asymmetric synthesis. A single contrast agent with ease of synthesis that works in multiple cancer types and simultaneously allows presurgical imaging, intraoperative deep-tissue three-dimensional visualization, and high-speed microscopic visualization of tumor margins via spatiotemporally complementary modalities would be beneficial. Methods: Due to the lack of commercial availability and the absence of detailed synthesis and characterization, we proposed a facile and scalable synthesis pathway for the symmetric NIR water-soluble heptamethine sulfoindocyanine IRDye78. The synthesis can be accomplished in four steps from commercially-available building blocks. Its symmetric resonant structure avoided asymmetric synthesis problems while still preserving the benefits of analogous IRDye800cw with commensurable optical properties. Next, we introduced a low-molecular-weight protein alpha-lactalbumin (α-LA) as the carrier that effectively modulates the hepatic clearance of IRDye78 into the preferred renal excretion pathway. We further implemented 89Zr radiolabeling onto the protein scaffold for positron emission tomography (PET). The multimodal imaging capability of the fluorophore-protein complex was validated in breast cancer and glioblastoma. Results: The scalable synthesis resulted in high chemical yields, typically 95% yield in the final step of the chloro dye. Chemical structures of intermediates and the final fluorophore were confirmed. Asymmetric IRDye78 exhibited comparable optical features as symmetric IRDye800cw. Its well-balanced quantum yield affords concurrent dual fluorescence and optoacoustic contrast without self-quenching nor concentration-dependent absorption. The NHS ester functionality modulates efficient covalent coupling to reactive side-chain amines to the protein carrier, along with desferrioxamine (DFO) for stable radiolabeling of 89Zr. The fluorophore-protein complex advantageously shifted the biodistribution and can be effectively cleared through the urinary pathway. The agent accumulates in tumors and enables triple-modal visualization in mouse xenograft models of both breast and brain cancers. Conclusion: This study described in detail a generalized strategic modulation of clearance routes towards the favorable renal clearance, via the introduction of α-LA. IRDye78 as a feasible alternative of IRDye800cw currently in clinical phases was proposed with a facile synthesis and fully characterized for the first time. This fluorophore-protein complex with stable radiolabeling should have great potential for clinical translation where it could enable an elegant workflow from preoperative planning to intraoperative deep tissue and high-resolution image-guided resection.

Gold/alpha-lactalbumin nanoprobes for the imaging and treatment of breast cancer.

Theranostic agents should ideally be renally cleared and biodegradable. Here, we report the synthesis, characterization and theranostic applications of fluorescent ultrasmall gold quantum clusters that are stabilized by the milk metalloprotein alpha-lactalbumin. We synthesized three types of these nanoprobes that together display fluorescence across the visible and near-infrared spectra when excited at a single wavelength through optical colour coding. In live tumour-bearing mice, the near-infrared nanoprobe generates contrast for fluorescence, X-ray computed tomography and magnetic resonance imaging, and exhibits long circulation times, low accumulation in the reticuloendothelial system, sustained tumour retention, insignificant toxicity and renal clearance. An intravenously administrated near-infrared nanoprobe with a large Stokes shift facilitated the detection and image-guided resection of breast tumours in vivo using a smartphone with modified optics. Moreover, the partially unfolded structure of alpha-lactalbumin in the nanoprobe helps with the formation of an anti-cancer lipoprotein complex with oleic acid that triggers the inhibition of the MAPK and PI3K-AKT pathways, immunogenic cell death and the recruitment of infiltrating macrophages. The biodegradability and safety profile of the nanoprobes make them suitable for the systemic detection and localized treatment of cancer.

Non-invasive In Vivo Imaging of Cancer Using Surface-Enhanced Spatially Offset Raman Spectroscopy (SESORS).

Rationale: The goal of imaging tumors at depth with high sensitivity and specificity represents a significant challenge in the field of biomedical optical imaging. 'Surface enhanced Raman scattering' (SERS) nanoparticles (NPs) have been employed as image contrast agents and can be used to specifically target cells in vivo. By tracking their unique "fingerprint" spectra, it becomes possible to determine their precise location. However, while the detection of SERS NPs is very sensitive and specific, conventional Raman spectroscopy imaging devices are limited in their inability to probe through tissue depths of more than a few millimetres, due to scattering and absorption of photons by biological tissues. Here, we combine the use of "Spatially Offset Raman spectroscopy" (SORS) with that of "surface-enhanced resonance Raman spectroscopy" (SERRS) in a technique known as "surface enhanced spatially offset resonance Raman spectroscopy" (SESO(R)RS) to image deep-seated glioblastoma multiforme (GBM) tumors in vivo in mice through the intact skull. Methods: A SORS imaging system was built in-house. Proof of concept SORS imaging was achieved using a PTFE-skull-tissue phantom. Imaging of GBMs in the RCAS-PDGF/N-tva transgenic mouse model was achieved through the use of gold nanostars functionalized with a resonant Raman reporter to create SERRS nanostars. These were then encapsulated in a thin silica shell and functionalized with a cyclic-RGDyK peptide to yield integrin-targeting SERRS nanostars. Non-invasive in vivo SORS image acquisition of the integrin-targeted nanostars was then performed in living mice under general anesthesia. Conventional non-SORS imaging was used as a direct comparison. Results: Using a low power density laser, GBMs were imaged via SESORRS in mice (n = 5) and confirmed using MRI and histopathology. The results demonstrate that via utilization of the SORS approach, it is possible to acquire clear and distinct Raman spectra from deep-seated GBMs in mice in vivo through the skull. SESORRS images generated using classical least squares outlined the tumors with high precision as confirmed via MRI and histology. Unlike SESORRS, conventional Raman imaging of the same areas did not provide a clear delineation of the tumor. Conclusion: To the best of our knowledge this is the first report of in vivo SESO(R)RS imaging. In a relevant brain tumor mouse model we demonstrate that this technique can overcome the limitations of conventional Raman imaging with regards to penetration depth. This work therefore represents a significant step forward in the potential clinical translation of SERRS nanoparticles for high precision cancer imaging.

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two 'flavors' of such nanoprobes: an antibody-functionalized one that targets the folate receptor - overexpressed in many ovarian cancers - and a non-targeted control nanoprobe, with distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity - which can be done conveniently during the surgical procedure - can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.