High throughput clogging free microfluidic particle filter by femtosecond laser micromachining.

In recent decades, driven by the needs of industry and medicine, researchers have been investigating how to remove carefully from the main flow microscopic particles or clusters of them. Among all the approaches proposed, crossflow filtration is one of the most attractive as it provides a non-destructive, label-free and in-flow sorting method. In general, the separation performance shows capture and separation efficiencies ranging from 70% up to 100%. However, the maximum flow rate achievable (µL/min) is still orders of magnitude away from those suitable for clinical or industrial applications mainly due to the low stiffness of the materials typically used. In this work, we propose an innovative hydrodynamic-crossflow hybrid filter geometry, buried in a fused silica substrate by means of the femtosecond laser irradiation followed by chemical etching technique. The material high stiffness combined with the accuracy of our manufacturing technique allows the 3D fabrication of non-deformable channels with higher aspect ratio posts, while keeping the overall device dimensions compact. The filter performance has been validated through experiments with both Newtonian (water-based solution of microbeads) and non-Newtonian fluids (blood), achieving separation efficiencies of up to 94% and large particles recovery rates of 100%, even at very high flow rates (mL/h).

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines.

BACKGROUND: Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy. RESULTS: In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression. CONCLUSIONS: Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome.

Application of Raman spectroscopy to the evaluation of F-actin changes in sea urchin eggs at fertilization.

The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

SERS Quantification of Galunisertib Delivery in Colorectal Cancer Cells by Plasmonic-Assisted Diatomite Nanoparticles.

The small molecule Galunisertib (LY2157299, LY) shows multiple anticancer activities blocking the transforming growth factor-ß1 receptor, responsible for the epithelial-to-mesenchymal transition (EMT) by which colorectal cancer (CRC) cells acquire migratory and metastatic capacities. However, frequent dosing of LY can produce highly toxic metabolites. Alternative strategies to reduce drug side effects can rely on nanoscale drug delivery systems that have led to a medical revolution in the treatment of cancer, improving drug efficacy and lowering drug toxicity. Here, a hybrid nanosystem (DNP-AuNPs-LY@Gel) made of a porous diatomite nanoparticle decorated with plasmonic gold nanoparticles, in which LY is retained by a gelatin shell, is proposed. The multifunctional capability of the nanosystem is demonstrated by investigating the efficient LY delivery, the enhanced EMT reversion in CRCs and the intracellular quantification of drug release with a sub-femtogram resolution by surface-enhanced Raman spectroscopy (SERS). The LY release trigger is the pH sensitivity of the gelatin shell to the CRC acidic microenvironment. The drug release is real-time monitored at single-cell level by analyzing the SERS signals of LY in CRC cells. The higher efficiency of LY delivered by the DNP-AuNPs-LY@Gel complex paves the way to an alternative strategy for lowering drug dosing and consequent side effects.

Raman Microscopy: Progress in Research on Cancer Cell Sensing.

In the last decade, Raman Spectroscopy (RS) was demonstrated to be a label-free, non-invasive and non-destructive optical spectroscopy allowing the improvement in diagnostic accuracy in cancer and analytical assessment for cell sensing. This review discusses how Raman spectra can lead to a deeper molecular understanding of the biochemical changes in cancer cells in comparison to non-cancer cells, analyzing two key examples, leukemia and breast cancer. The reported Raman results provide information on cancer progression and allow the identification, classification, and follow-up after chemotherapy treatments of the cancer cells from the liquid biopsy. The key obstacles for RS applications in cancer cell diagnosis, including quality, objectivity, number of cells and velocity of the analysis, are considered. The use of multivariant analysis, such as principal component analysis (PCA) and linear discriminate analysis (LDA), for an automatic and objective assessment without any specialized knowledge of spectroscopy is presented. Raman imaging for cancer cell mapping is shown and its advantages for routine clinical pathology practice and live cell imaging, compared to single-point spectral analysis, are debated. Additionally, the combination of RS with microfluidic devices and high-throughput screening for improving the velocity and the number of cells analyzed are also discussed. Finally, the combination of the Raman microscopy (RM) with other imaging modalities, for complete visualization and characterization of the cells, is described.

Resistance and Raman spectroscopy analysis of Parageobacillus thermantarcticus spores after γ-ray exposure.

Spores of the genus Bacillus are able to resist ionizing radiations and therefore they are a suitable biological model for studies in Astrobiology, i.e. the multidisciplinary approach to the study of the origin and evolution of life on Earth and in the universe. The resistance to γ-radiation is an important issue in Astrobiology in relation to the search for bacterial species that could adapt to life in space. This study investigates the resistance of spores of the thermophilic bacteria Parageobacillus thermantarcticus to γ-rays. The analysis of spores' response to irradiation at a molecular level is performed by means of Raman spectroscopy that allows to get insights in the sequence of events taking place during inactivation. The role of the γ-rays' dose in the inactivation of spores is also investigated, allowing to highlight the mechanism(s) of inactivation including DNA damage, protein denaturation and calcium dipicolinate levels.

Gold decorated porous biosilica nanodevices for advanced medicine.

Diatomite is a fossil material made of amorphous porous silica. In this work, polyethylene glycol (PEG)-modified diatomite NPs (PEG-DNPs) are decorated with gold NPs (AuNPs) by one-pot liquid-phase synthesis. Nanocomplexes (PEG-DNPs@AuNPs), with an average size of about 450 nm, are characterized by dynamic light scattering, electron microscopy, nitrogen adsorption/desorption analysis, UV-vis and photoluminescence spectroscopies. Preliminary studies on the use of the nanocomplex in nanomedicine are also presented. Tests performed incubating PEG-DNPs@AuNPs in physiological conditions reveal a good stability of material. Cellular uptake of labeled PEG-DNPs@AuNPs is investigated by confocal microscopy after incubation with human cervix epithelioid carcinoma (HeLa) cells up to 48 h: an efficient cytoplasmic localization is observed. In vitro cytotoxicity of nanocomplexes with a concentration up to 400 µg ml-1 for 72 h is also evaluated. The results suggest the use of PEG-DNPs@AuNPs as advanced nanodevices adding imaging features to the nanocomplexes, due to AuNPs as contrast agent.

Nanosphere Lithography on Fiber: Towards Engineered Lab-On-Fiber SERS Optrodes.

In this paper we report on the engineering of repeatable surface enhanced Raman scattering (SERS) optical fiber sensor devices (optrodes), as realized through nanosphere lithography. The Lab-on-Fiber SERS optrode consists of polystyrene nanospheres in a close-packed arrays configuration covered by a thin film of gold on the optical fiber tip. The SERS surfaces were fabricated by using a nanosphere lithography approach that is already demonstrated as able to produce highly repeatable patterns on the fiber tip. In order to engineer and optimize the SERS probes, we first evaluated and compared the SERS performances in terms of Enhancement Factor (EF) pertaining to different patterns with different nanosphere diameters and gold thicknesses. To this aim, the EF of SERS surfaces with a pitch of 500, 750 and 1000 nm, and gold films of 20, 30 and 40 nm have been retrieved, adopting the SERS signal of a monolayer of biphenyl-4-thiol (BPT) as a reliable benchmark. The analysis allowed us to identify of the most promising SERS platform: for the samples with nanospheres diameter of 500 nm and gold thickness of 30 nm, we measured values of EF of 4 × 105, which is comparable with state-of-the-art SERS EF achievable with highly performing colloidal gold nanoparticles. The reproducibility of the SERS enhancement was thoroughly evaluated. In particular, the SERS intensity revealed intra-sample (i.e., between different spatial regions of a selected substrate) and inter-sample (i.e., between regions of different substrates) repeatability, with a relative standard deviation lower than 9 and 15%, respectively. Finally, in order to determine the most suitable optical fiber probe, in terms of excitation/collection efficiency and Raman background, we selected several commercially available optical fibers and tested them with a BPT solution used as benchmark. A fiber probe with a pure silica core of 200 µm diameter and high numerical aperture (i.e., 0.5) was found to be the most promising fiber platform, providing the best trade-off between high excitation/collection efficiency and low background. This work, thus, poses the basis for realizing reproducible and engineered Lab-on-Fiber SERS optrodes for in-situ trace detection directed toward highly advanced in vivo sensing.

Modulated Raman Spectroscopy for Enhanced Cancer Diagnosis at the Cellular Level.

Raman spectroscopy is emerging as a promising and novel biophotonics tool for non-invasive, real-time diagnosis of tissue and cell abnormalities. However, the presence of a strong fluorescence background is a key issue that can detract from the use of Raman spectroscopy in routine clinical care. The review summarizes the state-of-the-art methods to remove the fluorescence background and explores recent achievements to address this issue obtained with modulated Raman spectroscopy. This innovative approach can be used to extract the Raman spectral component from the fluorescence background and improve the quality of the Raman signal. We describe the potential of modulated Raman spectroscopy as a rapid, inexpensive and accurate clinical tool to detect the presence of bladder cancer cells. Finally, in a broader context, we show how this approach can greatly enhance the sensitivity of integrated Raman spectroscopy and microfluidic systems, opening new prospects for portable higher throughput Raman cell sorting.

Towards a New Generation of Hormone Therapies: Design, Synthesis and Biological Evaluation of Novel 1,2,3-Triazoles as Estrogen-Positive Breast Cancer Therapeutics and Non-Steroidal Aromatase Inhibitors.

Aromatase inhibitors (AIs) show promising features as drugs to treat estrogen-responsive breast cancer as they block aromatase activity, the key enzyme in estrogen synthesis. The current AIs approved by the Food and Drug Administration for breast cancer treatment present severe adverse effects. For these reasons, it is important to develop of new AIs that are more specific and sensitive. In this paper, we report the synthesis and the characterization of new nonsteroidal aromatase AIs containing triazoles moieties for the treatment of hormone-dependent breast cancer in post-menopausal women. A new series of 1,2,3-triazole based molecules were successfully synthetized and their chemical structures were determined from the spectral data (FT-IR, 13C NMR, 1H NMR, mass spectroscopy) and micro-analytical data. Additionally, the physical properties of the newly synthesized derivatives were reported. The novel compounds were also tested for their anticancer activity in both breast cancer (MCF7 and T-47D) and normal breast (MCF 10A) cell lines, evaluating their effect on cell proliferation, migration, and invasion. The results revealed that the compounds exhibited promising and specific anti-cancer action.

Cancer metabolic features allow discrimination of tumor from white blood cells by label-free multimodal optical imaging.

Circulating tumor cells (CTCs) are tumor cells that have penetrated the circulatory system preserving tumor properties and heterogeneity. Detection and characterization of CTCs has high potential clinical values and many technologies have been developed for CTC identification. These approaches remain challenged by the extraordinary rarity of CTCs and the difficulty of efficiently distinguishing cancer from the much larger number of white blood cells in the bloodstream. Consequently, there is still a need for efficient and rapid methods to capture the broad spectrum of tumor cells circulating in the blood. Herein, we exploit the peculiarities of cancer metabolism for discriminating cancer from WBCs. Using deuterated glucose and Raman microscopy we show that a) the known ability of cancer cells to take up glucose at greatly increased rates compared to non-cancer cells results in the lipid generation and accumulation into lipid droplets and, b) by contrast, leukocytes do not appear to generate visible LDs. The difference in LD abundance is such that it provides a reliable parameter for distinguishing cancer from blood cells. For LD sensitive detections in a cell at rates suitable for screening purposes, we test a polarization-sensitive digital holographic imaging (PSDHI) technique that detects the birefringent properties of the LDs. By using polarization-sensitive digital holographic imaging, cancer cells (prostate cancer, PC3 and hepatocarcinoma cells, HepG2) can be rapidly discriminated from leukocytes with reliability close to 100%. The combined Raman and PSDHI microscopy platform lays the foundations for the future development of a new label-free, simple and universally applicable cancer cells' isolation method.

Profilin 1 deficiency drives mitotic defects and reduces genome stability.

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

Diatom biosilica in plasmonics: applications in sensing, diagnostics and therapeutics [Invited].

Several living organisms are able to synthesize complex nanostructures provided with peculiar physical and chemical properties by means of finely-tuned, genetically controlled biomineralization processes. Frustules, in particular, are micro- and nano-structured silica shells produced by ubiquitous diatom microalgae, whose optical properties have been recently exploited in photonics, solar energy harvesting, and biosensing. Metallization of diatom biosilica, both in the shape of intact frustules or diatomite particles, can trigger plasmonic effects that in turn can find application in high-sensitive detection platforms, allowing to obtain effective nanosensors at low cost and on a large scale. The aim of the present review article is to provide a wide, complete overview on the main metallization techniques applied to diatom biosilica and on the principal applications of diatom-based plasmonic devices mainly but not exclusively in the fields of biochemical sensing, diagnostics and therapeutics.

Label-free probing of G-quadruplex formation by surface-enhanced Raman scattering.

In this work, we establish the use of surface-enhanced Raman scattering (SERS) as a label-free analytical technique for the direct detection of G-quadruplex formation. In particular, we demonstrate that SERS analysis allows the evaluation of the relative stability of G quadruplexes that differ for the number of G tetrads and investigate several structural features of quadruplexes, such as the orientation of glycosidic bonds, the identification of distortions in the sugar-phosphate backbone, and the degree of hydrogen-bond solvation. Herein, the fluctuation of the SERS spectra, due to the specific interaction of vibrational modes with the SERS-active substrate, is quantitatively analyzed before and after quadruplex formation. The results of this study suggest a perpendicular orientation of the quadruplexes (with or without the 3'-tetra end linker) with respect to the silver colloidal surface, which opens new perspectives for the use of SERS as a label-free analytical tool for the study of the binding mode between quadruplexes and their ligands.

Biosensing Using SERS Active Gold Nanostructures.

Surface-enhanced Raman spectroscopy (SERS) has become a powerful tool for biosensing applications owing to its fingerprint recognition, high sensitivity, multiplex detection, and biocompatibility. This review provides an overview of the most significant aspects of SERS for biomedical and biosensing applications. We first introduced the mechanisms at the basis of the SERS amplifications: electromagnetic and chemical enhancement. We then illustrated several types of substrates and fabrication methods, with a focus on gold-based nanostructures. We further analyzed the relevant factors for the characterization of the SERS sensor performances, including sensitivity, reproducibility, stability, sensor configuration (direct or indirect), and nanotoxicity. Finally, a representative selection of applications in the biomedical field is provided.

Interaction of nanoparticles with endotoxin Importance in nanosafety testing and exploitation for endotoxin binding.

The interaction between engineered nanoparticles and the bacterial lipopolysaccharide, or endotoxin, is an event that warrants attention. Endotoxin is one of the most potent stimulators of inflammation and immune reactions in human beings, and is a very common contaminant in research labs. In nanotoxicology and nanomedicine, the presence of endotoxin on the nanoparticle surface affects their biological properties leading to misinterpretation of results. This review discusses the importance of detecting the endotoxin contamination on nanoparticles, focusing on the current method of endotoxin detection and their suitability for nanoparticulate materials. Conversely, the capacity of nanoparticles to bind endotoxin can be enhanced by functionalization with endotoxin-capturing molecules, opening the way to the development of novel endotoxin detection assays.

SERS Sensing of Bacterial Endotoxin on Gold Nanoparticles.

Engineered gold nanoparticles (AuNPs) find application in several fields related to human activities (i.e., food and cosmetic industry or water purification) including medicine, where they are employed for diagnosis, drug delivery and cancer therapy. As for any material/reagent for human use, the safety of AuNPs needs accurate evaluation. AuNPs are prone to contamination by bacterial endotoxin (lipopolysaccharide, LPS), a potent elicitor of inflammatory responses in mammals. It is therefore important, when assessing AuNP immunosafety and immune-related effects, to discriminate between inflammatory effects intrinsic to the NPs from those caused by an undeliberate and undetected LPS contamination. Detection of LPS contamination in AuNP preparations poses different problems when using the current LPS detection assays, given the general interference of NPs, similar to other particulate agents, with the assay reagents and endpoints. This leads to time-consuming search for optimal assay conditions for every NP batch, with unpredictable results, and to the use in parallel of different assays, each with its weaknesses and unpredictability. Thus, the development of highly sensitive, quantitative and accurate assays able to detect of LPS on AuNPs is very important, in view of their medical applications. Surface-enhanced Raman spectroscopy (SERS) is a label-free, sensitive, chemical-specific, nondestructive and fast technique that can be used to directly obtain molecular fingerprint information and a quantitative analysis of LPS adsorbed on AuNPs. Within this study, we describe the use of SERS for the label-free identification and quantitative evaluation - down to few attograms - of the LPS adsorbed on the surface of 50 nm AuNPs. We thus propose SERS as an efficient tool to detect LPS on the AuNP surface, and as the basis for the development of a new sensitive and specific LPS-detection sensor based on the use of AuNPs and SERS.

Interaction between Macrophages and Nanoparticles: In Vitro 3D Cultures for the Realistic Assessment of Inflammatory Activation and Modulation of Innate Memory.

Understanding the modes of interaction between human monocytes/macrophages and engineered nanoparticles is the basis for assessing particle safety, in terms of activation of innate/inflammatory reactions, and their possible exploitation for medical applications. In vitro assessment of nanoparticle-macrophage interaction allows for examining the response of primary human cells, but the conventional 2D cultures do not reproduce the three-dimensional spacing of a tissue and the interaction of macrophages with the extracellular tissue matrix, conditions that shape macrophage recognition capacity and reactivity. Here, we have compared traditional 2D cultures with cultures on a 3D collagen matrix for evaluating the capacity gold nanoparticles to induce monocyte activation and subsequent innate memory in human blood monocytes in comparison to bacterial LPS. Results show that monocytes react to stimuli almost in the same way in 2D and 3D cultures in terms of production of TNFα and IL-6, but that notable differences are found when IL-8 and IL-1Ra are examined, in particular in the recall/memory response of primed cells to a second stimulation, with the 3D cultures showing cell activation and memory effects of nanoparticles better. In addition, the response variations in monocytes/macrophages from different donors point towards a personalized assessment of the nanoparticle effects on macrophage activation.

Innate Memory Reprogramming by Gold Nanoparticles Depends on the Microbial Agents That Induce Memory.

Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs. particulate form (MDP and the gram-positive bacteria Staphylococcus aureus; ß-glucan and the ß-glucan-producing fungi C. albicans), and as whole microrganisms that were either killed (S. aureus, C. albicans) or viable (the gram-negative bacteria Helicobacter pylori). The memory response was assessed in vitro, by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, ß-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori-induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures.

Online fluorescence suppression in modulated Raman spectroscopy.

Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.