Surface-enhanced Raman spectroscopy offers ultra-sensitive vibrational fingerprinting within biological cells. Surface-enhanced Raman spectrocopy (SERS) offers ultrasensitive vibrational fingerprinting at the nanoscale. Its non-destructive nature affords an ideal tool for interrogation of the intracellular environment, detecting the localisation of biomolecules, delivery and monitoring of therapeutics and for characterisation of complex cellular processes at the molecular level. Innovations in nanotechnology have produced a wide selection of novel, purpose-built plasmonic nanostructures capable of high SERS enhancement for intracellular probing while microfluidic technologies are being utilised to reproducibly synthesise nanoparticle (NP) probes at large scale and in high throughput. Sophisticated multivariate analysis techniques unlock the wealth of previously unattainable biomolecular information contained within large and multidimensional SERS datasets. Thus, with suitable combination of experimental techniques and analytics, SERS boasts enormous potential for cell based assays and to expand our understanding of the intracellular environment. In this review we trace the pathway to utilisation of nanomaterials for intracellular SERS. Thus we review and assess nanoparticle synthesis methods, their toxicity and cell interactions before presenting significant developments in intracellular SERS methodologies and how identified challenges can be addressed. Of particular interest are plasmonic nanoparticles (NPs), primarily of gold (Au) and silver (Ag), owing to their unique optical properties which allow intense scattering of light to achieve quantification, localisation and therefore imaging of biological systems The heightened intensities observed in SERS relative to spontaneous Raman spectroscopy are primarily due to the enhanced electric fields produced by conductance electrons at nanomaterial surfaces, which undergo collective oscillations known as surface plasmons. Combination of this electromagnetic mechanism with additional pathways such as charge transfer and chemisorption induced resonance Raman effects result in enhancement by factors of 106–1010 in SERS over spontaneous Raman spectroscopy. Such enhancement is crucial to studies of intact and living cells as the concentrations of biomolecules inside cells are typically of the order of nM. It allows fine spectral details to be observed without interference from the vibrational peaks of H2O observed in IR spectroscopy. (Surface-enhanced) Raman spectroscopy also proves advantageous as it is a non-destructive and label-free tool with simple or no preparation of samples, utilising an increased depth of penetration by NIR radiation. Currently, fluorescence imaging is commonplace and benefits as an intracellular technique from large intrinsic signals, availability of a wide range of labels  and the ability to tune the response of labels to analytes or pH. However, it lacks the specificity of information provided by SERS, as only a finite number of dyes can be simultaneously employed for probing the desired environment due to spectral overlap. Such tagging of molecules can also perturb the natural, molecular-level progression of biological pathways being analysed. It is worth noting that prolonged exposure to nanoparticles can also play an active role in mediating biological effects. However, fluorescence has further limitations that signals get photobleached over time compared to Raman-based techniques. Given that SERS has been shown to possess single molecule sensitivity and can be comparable or more sensitive than fluorescence for biological assays it offers numerous advantages and complimentary information for intracellular analysis.Surface-enhanced Raman spectroscopy (SERS), potentiated by noble metal nanostructures, was first observed in 1973 and subsequently verified in 1977, when the spontaneous Raman signal of adsorbed pyridine was easily measured at a roughened silver electrode.in cellulo studies, particle diameter must be small enough to penetrate the intracellular matrix yet larger than 15 nm to achieve SERS enhancement. Spherical AgNPs exhibit stronger plasmonic fields than those of Au, especially in the visible region of the electromagnetic spectrum owing to the partial Au plasmon band overlap with its interband electronic transitions. Notwithstanding this, AuNPs are more widely applied in biological studies due to their well established and controlled methods of synthesis along with good biocompatibility and chemical stability. The ability to track and detect plasmonic NPs using various analytical tools, especially their localised surface plasmon resonance bands, which can be synthetically tuned into the near infrared region , is an added advantage. Facile surface chemistry allows for easy surface functionalisation, affording not only the binding of specific delivery peptides, but also other applications such as artificial antibodies with binding affinities precisely tuned by varying the density of surface bound ligands. The ability to shield unstable drugs or poorly soluble imaging contrast agents to facilitate their delivery to otherwise inaccessible regions of the body is augmented by AuNPs’ multivalent nature.For successful cellular investigations by SERS, however, the selection of suitable NPs is essential, which must overcome issues such as internalisation and toxicity while maintaining desired optical properties. For From the above it can thus be seen that the type of nanoparticles, choice of their surface chemistry and consequent interaction with cells  can be critical to their utilisation for intracellular SERS. Through this review we therefore aim to provide an insight into all aspects involving intracellular SERS sensing and imaging. We trace all the steps involved in the use of nanostructures for intracellular SERS, starting with an evaluation of the main synthetic methods and different types of nanoparticles. While spherical AuNPs are currently the most widely used and understood, AgNPs have been used and alternative structures are becoming popular and will be discussed where relevant. This will be followed by an overview of crucial NP–cell interactions. Physical and diffusive routes of particle internalisation are examined along with biocompatibility factors such as tunability of surface properties for internalisation and resulting toxicity. The two prevalent approaches, the SERS-reporter and reporter-free approaches, to intracellular SERS are then presented. State-of-the-art and recent significant studies are discussed and the potential of intracellular SERS for numerous applications in life sciences, therapeutics and drug discovery is discussed. However, it was Michael Faraday's recognition that the properties of pure colloidal (or ‘activated’) Au, following its synthesis by reduction of Au chloride by phosphorus in the presence of carbon disulphide as a stabiliser, were due to its minute size, which attracted renewed scientific attention. With the wide availability of nanoscale characterisation tools, the design of AuNPs with precisely controlled sizes and shapes has since been comprehensively developed.The earliest description of obtaining colloidal Au can be traced to ancient Chinese, Arabian, and Indian treatises dating back to as early as 4–5th BCE wherein it was utilised mostly for medicinal purposes.via a citrate-mediated growth method, which was first demonstrated by Turkevich et al. in 1951  and later systematically studied and improved by Frens  with high aspect ratios, nanocages with a hollow interior and top down deposited nanochips. The size of AuNPs can also be precisely tuned from a few to hundreds of nm.y Frens . A “neckditions . This meet al. developed a sequential growth method for achieving better control over monodispersity of both the silica core and the Au nanoshell with both high purity and high yield. The ultrasmall (1–2 nm in diameter) AuNPs were first anchored onto silica NP cores, followed by further reduction to form a complete Au shell layer  shell of silica or alumina, preventing uncontrolled AuNP aggregation while retaining strong optical enhancement. In addition, the chemically inert and fully enclosed shell layer allows particles to adjust its conformation to the diverse contours of non-flat samples such as single-crystal edges and biological cells. Au nanocages developed by Xia et al. possess unique cargo-holding hollow structures in addition to attractive optical properties, thus are useful for many biomedical applications, including bioimaging, cancer diagnosis, photothermal therapy, and drug delivery  with multiple steps and poor inter-batch reproducibility. Simply scaling up the reactant volumes does not necessarily result in the same reaction scheme, because nucleation and growth of colloids are very complex processes that are extremely sensitive to experimental conditions. Implementing continuous and automated NP production processes is therefore desirable to facilitate scale up and improve homogeneity.et al. showed that by using microfluidic droplet reactors, continuous-flow mass production of a number of noble-metal NPs with controlled sizes and shapes can be achieved. In this method, microlitre-sized droplets containing various reagents were continuously generated in a fluidic device fabricated by assembling silica capillaries inside polymer tubes. Microfluidic techniques are operated at a steady state, offering superior control over reaction conditions such as reagent addition, mixing, reaction time and temperature. NP size depends upon relative reaction and mixing rates along with the extent of polydispersity, enhanced by the improved control over heat and mass transport which accompanies the technology. The approach also lends itself to accurate and rapid screening of conditions for quality control purposes.Xia  using a previously established ‘polyol process’. This reduction method of ionic, inorganic precursors dissolved in liquid polyols  utilises microwave irradiation to achieve more uniform heating, nucleation control and increased reaction rates. Thus AgNPs could be produced from a silver acetate substrate within a matter of seconds. The penetration depth of microwaves (a few centimetres) renders the scale-up of batch production impractical yet is still large enough to rationally design a continuous-flow synthesis system with high yields.Microfluidic AgNP synthesis has been performed in a continuous flow single-mode microwave reactorSimilarly, hollow gold nanoparticles (HGNPs) particularly require strict dimensional control within large-scale production, unachievable by batch processes. This is because the ratio of shell diameter to thickness determines the peak position of its surface plasmon resonance (SPR), while uniformity controls SPR bandwidth. High quality HGNPs were produced from galvanic replacement of sacrificial Co templates, including stages for poly-ethylene glycol (PEG) functionalisation and sterilisation on a singular platform with rational use of reactants and no need for time consuming purification steps by the scheme depicted in et al. utilises extract from commercially available Illicium verum  in an effective one-step and economical synthesis of biocompatible AuNPs at room temperature. Although boiling is required for extraction, biologically synthesised AuNPs exhibited 20× and 5× fold toxicity reduction in A549 cells compared to those produced synthetically via sodium borohydrate and citrate reduction mechanisms respectively. This was attributed to the protective capping of NPs by polyphenols present in SA extract, with bio-AuNP exposed cells displaying reduced caspase levels, a key protein in the mechanism of apoptosis. AuNPs have been biosynthesised using the fruit extract of Couroupita guianensis  as a bio-reducing agent of Au3+ to yield anisotropic face centred cubic crystalline structured particles of 26 ± 11 nm diameter, with high stability and purity. These NPs also possessed extraordinary antioxidant properties, as revealed by in vitro assays, and free radical scavenging capabilities making them ideally suited to nanomedicine. In a similar vein, AgNPs have also been extracellularly synthesised without surfactant or external energy by mixing silver solution with extracts from Azadiracta indica and Zingiber officiale (Neem and Ginger respectively). This synthesis produced physiologically stable, highly biocompatible AgNPs within 1–2 h compared to the 2–4 days required by microorganisms in in vivo NP production. Biosynthesised NPs produced by Potara et al. were used to probe C26 murine colon carcinoma cells in vitro to reveal subcellular components and the localisation of the AgNPs by confocal Raman spectroscopy and K-means clustering analysis. Further to the examples provided, a comprehensive review of green NP sources and their significance to cancer management and developing anti-cancer stem cell therapeutics through enhanced biocompatibility is presented by the Sreelekha and coworkers. As with traditional synthetic methods, the major limitation of biosyntheses lies in their batch production format, presenting difficulties in upscaling production. A lack of inter-batch reproducibility was also reported as a result of natural variation in the polyphenol content of SA, despite minimal variation in toxicity within each batch of product. Therefore, the classical trade-off between sustainable and large-scale industrial synthesis is also applicable to NP production, thus requiring further development.Aside from the capability for scaling-up, modern synthetic chemistry is required to be sustainable. Another ‘green’ synthetic route for AuNP production lies in biosynthesis, with methods not requiring the high-energy input of irradiation based methods. An approach presented by Sathishkumar in vivo cells, as mechanistic understanding of how physiochemical properties of NPs affect NP/cellular interactions is limited. Although AuNPs are generally considered non-toxic to live cells (discussed later), evidence exists that their prolonged presence within the intracellular matrix does perturb their biology and induces stress. SERS has been carried out using glass capillary probes decorated with AuNPs to detect proteins or metabolites both intra- and extracellularly to overcome this issue. This review however focusses on the more widely applied NP-based intracellular SERS approach, in which NPs can be applied to samples as a probe reagent for internalisation at the time of analysis so as to minimise the effects of prolonged exposure on cellular pathways and thus yield sizeable advantages over conventional labelling techniques.The interaction between living cells and any exogenous material is one of great complexity. The cellular matrix is a vastly complicated environment which plays host to a range of internal structures and sensitive biochemical pathways, possessing variety in cargo uptake processes and transport vesicles. Therefore, it is with caution that NPs are introduced to living cells for developing models of healthy The key to successful utilisation of NPs inside cells for SERS and other measurements is understanding and manipulating their mechanism of uptake and cellular distribution. Internalisation methods generally fall into three categories: involuntary delivery by physical methods, passive diffusion, and active (or voluntary) uptake. Microinjection has been of particular interest in single cell studies, providing tight control of dosage and timing of delivery but is highly sensitive as inappropriate use  can easily damage cellular components. Single 100 nm AuNPs have been delivered to mammalian cell nuclei by combination of optical tweezing and opto-injection, along with using laser irradiation to also assist NP injection of 15–30 nm AuNPs. Despite the tight control offered, the complexity of both procedure and instrumentation limits the technique which is also confined to larger NPs. The major advantage of NP uptake by passive diffusion is that internalised cargos enter the cytosol directly and are not required to escape from vesicles involved in endocytosis. Colloidal semiconductor core/shell quantum dots coated with the zwitterionic thiol ligand d-penicillamine are effectively similar to globular proteins in size and surface charge, and are internalised by HeLa cells via clathrin-mediated endocytosis. However, these were also shown to enter erythrocytes by passive diffusion (given their lack of endocytotic machinery) without formation of holes in the lipid bilayer. AuNPs of diameter ≤200 nm have also been observed to penetrate the bilayer of erythrocytes.The basis of physical insertion methods is the theory of applying a force to the cell in order to create localised membrane pores and increase cell permeability. Electroporation and microinjection achieve this with the application of electrical and physical force respectively. The TATHA2 is 1 to 20 amino acid sequence of the influenza A virus hemagglutinin protein (HA2) connected to a 10 amino acid cell permeable HIV Trans-Activator of Transcription (TAT) protein transduction domain (PTD). The TAT PTD binds to the cell surface and penetrates the membrane while the pH sensitive lipid membrane destabilising sequence of the HA2 domain facilitates endosomal escape and transduction of the fusion peptide. In the above work by Bhardwaj et al. it was found that although yeast cells can tolerate high electroporation doses, severe damage was observed with the presence of AgNPs. Free diffusion of AgNPs resulted in poor uptake, internalisation and endosome entrapment whereas these parameters were rapid and large with TATHA2-AgNPs. A uniform intracellular distribution was also observed, which is a requirement for detection of ubiquitously distributed molecules in cell based biosensors. Conjugation of cell penetrating peptides (CPP) such as arginylglyclaspartic (RGD) or nuclear localisation signal peptides (NLS) to NPs also facilitate cellular uptake, as well as help guide probes out of the endolysosomal pathway as discussed under the section on ‘Manipulating interactions’.The uptake mechanism of AgNPs in yeast cells was recently investigated, comparing entry by electroporation with diffusive approaches by TATHA2 peptide functionalised (facilitated) and citrate capped AgNPs for uptake by endocytosis.via endocytosis is by far the most commonly used internalisation strategy, whereby metallic species are taken up by the cell's intrinsic machinery. This is owing to the method's relative simplicity and minimal sample preparation as well as tunability by appropriate surface modification.Voluntary uptake of particles  For macromolecules and proteins which are too large to pass directly through the plasma membrane, the cell possesses different mechanisms for their intake from the environment. These intake mechanisms are generally referred to as endocytosis, the commencement of the vesicular endolysosomal pathway through the cell  as well as the specific cell type. Broadly, these pathways are divided into phagocytosis, macropinocytosis, endocytosis via non-coated vesicles and energy-dependent, receptor-mediated endocytosis . The receptor is initially internalised with the ligand and is gradually recycled to the plasma membrane  along with various macromolecules from other intracellular, catabolic processes and due to membrane turnover. These include molecules incorporated previously through autophagy. During the lysosomal pathway, most of the molecular digestion and degradation takes place while the pH  in these vesicles becomes more and more acidic. Subsequent dynamic changes of the molecular composition and the activity of enzymes inside the vesicles also result in modification of the encapsulated molecules. At the end of the lysosomal pathway, vesicles with indigestible material become residual bodies and are secreted from the cell via exocytosis. Different maturation and fusion steps within this whole pathway are colour-indicated in Early endosomes are initially relatively small, have a mildly acidic pH and are known to be the main sorting station in the endosomal pathway. While intravesicular fluids are recycled, early endosomes fuse with other incoming cargos from different internalisation pathways . With the intention to carry out intracellular SERS experimentation, several factors contribute to identifying the optimal NP diameter. AuNPs larger than 15 nm have a sufficiently large scattering cross-section and exhibit plasmonic activity to provide the required optical contrast and electric field enhancement, which must be considered when utilizing optical based analytical tools for the localization of AuNPs inside cells.via endocytotic pathways is to facilitate the diffusion by employing surface modification. Following production by previously discussed synthetic methods, there is a common requirement for stabilisation in order to prevent further growth or aggregation. Charged ligands such as citrate are often utilised for this purpose as coulombic repulsion serves to keep produced NPs separate. However, the negative charge possessed by citrate and other ligands is unfavourable for interaction with the similarly negatively charged cell membrane upon nanoparticle feeding, a process which rather favours small uncharged cargos. In solution-based conditions, macromolecules such as serum proteins adhere to the noble metal surface by physical or chemical adsorption to produce the double layer structure displayed in via intermolecular forces such as protein–protein interaction which shields the inner layer from the particle environment. Despite this, the make-up of the protein corona is subject to dynamic changes in composition and thickness  with adsorbed proteins affecting the hydrodynamic diameter of an NP. Initial formation of the corona occurs rapidly upon contact and in the case of culture medium, a stable and biocompatible structure is formed. This process is of huge synthetic advantage for functionalisation of nanoparticles to achieve facilitated uptake and cellular compartment targeting as even inner layer proteins can be substituted at the surface following a few hours’ incubation with desired ligands, requiring minimal sonication or heat input. This hard corona layer gives the nanoparticle its biochemical identity as experienced by the cell upon contact and remains relatively unaffected by biophysical events such as internalisation- therefore selection of ligand is crucial in achieving the desired uptake and localisation of a SERS nanoprobe. However, environmental changes such as enzymatic presence or the pH are mainly experienced by the outer corona layers, allowing for the use of organic molecules as SERS labels or attachment of fluorescent tags in (or as) the inner layer.A prominent mechanism for aiding cellular uptake  Cellular entry and escape from endolysosomal vesicles into the cell through the plasma membrane is associated with the ability to penetrate or pass through the lipid bilayer. Permeability of the lipid bilayers is known to be achieved by the interaction of cationic molecules with the negatively charged plasma membrane which is thereby disrupted and forms nanopores. The choice of molecules spans amine-containing polymers, polypeptides and cationic liposomes. PEGylation of NPs is a very common surface modification for aiding cellular internalisation, providing a ‘masking’ effect for the NPs to cells of the immune system. They are also more tolerant to salt aggregation than either citrate or CTAB coated NPs, with smaller cargos being less imposing to cell biology. An additional benefit is that PEG coating hydrophilises the NP surface to serve as a ligand for the attachment of drugs or genes for targeted delivery.As previously mentioned, surface modification of AgNPs by TATHA2 has achieved enhanced uptake by endocytosis in yeast cells. protein transduction domains, CPPs and ligands. Rhim and co-workers have demonstrated the cytoplasmic localisation of AuNPs conjugated to cationic liposomes alongside plasmid DNA resulting in an improved efficiency of cellular transfection. This is thought to be achieved by charge neutralisation by liposomes of the plasmid–AuNP complex which results in a more favourable interaction with the plasma membrane.Once internalised, a wide range of biochemical delivery strategies are known for evading endolysosomal vesicles. Typically, they are based on the conjugation of the particle surface with various types of molecules such as cationic molecules, Even though the exact mechanism of its interaction with biological membranes is not yet known, TAT is thought to mediate cellular import via multiple routes including direct membrane passage and intake through endocytosis.Furthermore, CPPs have widely been applied to target specific intracellular organelles. Many peptide sequences have been derived from natural systems used by viruses such as the TAT peptide derived from the HIV-1 virus which, conjugated to AuNPs, is known to allow cytoplasmic localisation.via RME. Furthermore, ligands have particularly been used to target selected cellular components such as the nucleus with attachment of nuclear localisation signal (NLS) peptides.Ligand molecules facilitate the directed interaction of the cargo with specific receptors by binding to them. This can be used for the delivery of AuNPs into cells as for instance shown by Tkachenko and co-workers who demonstrated that NPs modified with adenoviral RME peptides gain entry into cells in vitro cell model becomes less representative of the natural state. In general, cytotoxicity of NPs is attributed to the size of both single particles and clusters thereof, surface modifications, NP concentration and composition of the protein corona in addition to the cell type. Their size determines the crossing of lipid membranes by NPs into various subcellular structures possessing size exclusive pores. Confinement of NPs in cellular vesicles such as endosomes may however still present toxicity, when the local concentration of the metal far exceeds its LD50 . Smaller particles have high surface to volume ratio and therefore can have increased level of interactions with the environment. Indeed single particles of size less than 2 nm in diameter have been shown by several studies to have damaging effects on cells.A vital consideration to the intracellular SERS experiment is the effect of the administered NP on its host. That is, any change or decline in key cellular processes induced by particle–cell interactions decrease the validity of the investigation conducted, as the In addition to size based effects, homeostatic mechanisms are also disrupted by the presence of ‘foreign’ metal within the cell. Oxidative stress results from the production of reactive oxidative species (ROS) upon cellular recognition of foreign material and is usually compensated for by homeostatic defence mechanisms. However, at high metal concentrations these mechanisms are unable to maintain normal cell function, therefore cause toxicity and cell death. However, pretreatment of the 1.4 nm NPs with reducing agents/antioxidants N-acetylcysteine, glutathione, and TPPMS reduced the observed toxicity. AuNPs of similar size but capped with glutathione also induced no oxidative stress. Besides the size dependence, ligand chemistry was also therefore defined as a critical parameter for biocompatibility and toxicity of AuNPs.Surface modifications also impede upon cell viability. Oxidative stress, evidenced by compromise of mitochondrial potential, integrity and substrate reduction along with presence of ROS, was induced in HeLa cells by triphenylphosphine monosulfonate capped 1.4 nm AuNPs to show increased cytotoxicity relative to 15 nm AuNPs of similar chemistry. by the induction of oxidative stress to host cells, exhibiting protein misfolding, mitochondrial dysfunction and impaired DNA repair leading to cell death. This apparent toxicity can however be advantageous for applications in cancer therapy, with AgNPs shown to be toxic towards tumour cell lines HeLa and U937. In contrast, AuNPs are usually considered to be non-cytotoxic and inert with limited reports of adverse effects. Direct comparison of the two materials’ toxicity is complicated by the large number of factors described in this section, including the differing surface coatings required for stability.The material of the nanoparticle is bound to have an effect. For instance, AgNPs are frequently reported as toxic who studied the viability of microglia when incubated with spherical AuNPs, nanorods and nanourchins coated with either PEG or CTAB. No toxicity was associated with any variant at low culture medium NP concentrations (<9 × 109 NPs per mL), however at 9 × 109 NPs per mL cellular activity was reduced by 94% following exposure to CTAB coated nanospheres with no relative toxicity exhibited in the other CTAB particle shapes (slight toxicity in CTAB nanorods with 13% activity reduction at 1011 NPs per mL). The modest reduction in cell viability in Au nanospheres compared to Au NRs was likely due to the increased uptake rate of nanospheres. The finding that CTAB coated NPs bore a greater toxicity than those coated with PEG is consistent with numerous other studies. CTAB is used in synthesis as a shape-directing agent and stabilising ligand due to its positive charge, which forms strong interactions with the negatively charged plasma membrane. This disruption of membrane structure is responsible for CTAB's associated toxicity to cells at concentrations over ∼10 nM. Reduction of this toxicity can however be achieved by surface modification with other capping agents such as PEG, PAA, PAH  and polystyrene sulfonate.The effects of shape, surface chemistry and NP concentration have been reported by Hutter, Various shortcomings in toxicity assays for NP applications have been identified, including lack of complete characterisation of NPs utilised in experiments, inconsistent reporting of NP concentration , and a lack of standardisation of toxicology method parameters such as dose range and incubation media, all of which make comparisons between nanoparticles complicated. For instance, the aforementioned toxicity of CTAB has also been attributed to the free molecule's presence in AuNP solution, which raises the need for tight ‘supernatant control’ steps to be taken for valid NP toxicity evaluation. Additionally, NPs themselves may affect performance of routine toxicity assays, requiring validation in the presence of the specific type of NP employed as variation in structure or size may alter the performance differently. Current ambiguities arise from two predominant factors, the first being the complex governance of the phenomenon by a large number of both chemical and physical properties of the NP along with cell types and the degree of NP internalisation. The second being a lack of standardisation in experimental design, execution of procedure and data treatment.In spite of numerous toxicity studies, there remains a fundamental requirement for standardisation of investigative procedure before an acceptable consensus on AuNP toxicity can be reached.et al. gathered existing data from published studies conducting MTT assay of HeLa cells exposed to differently functionalised AuNPs, for analysis by the metric of numerical particle concentration as opposed to mass concentration or particle size. This produced a simplified view of how parameters such as NP size, concentration and surface coating impact individual cell viability and concluded that size dependence is much less important than the number of particles present per unit volume. Differently functionalised AuNPs behaved similarly, with the surface coating of the NPs defining the range of particle concentrations at which toxic effects commence.To illustrate the latter point, Fratoddi in vitro and beyond, however promising steps are being taken to achieve this.Thus overall, the requirement for standardisation of nanoparticle internalisation protocols and data treatment remains eminent for the progression of SERS experimentation on single cells et al. detected the presence of antitumor drugs doxorubicin (DOX) and 4′0-tetrahydropyranyl-adriamycin (THP-ADM) in the nucleus and cytoplasm of living cancer cells using citrate reduced AgNPs. Since its first intracellular utilisation, SERS has been successfully applied for detection of a vast array of biological markers and metabolites, in turn for sensing and tracking environmental changes through cellular pathways and processes. These include monitoring of cellular functions, dynamics, enzyme kinetics, stress response, apoptosis and cell death along with probing specific compartments such as the mitochondria and tracking of drugs released into the cytoplasm by NP carriers.The first application of SERS within cells was carried out in 1991 when Nabiev The cell-based SERS experiment generally takes one of the following two methodologies: the SERS-reporter (SERS-label) approach or the reporter-free (label-free) SERS approach. While the SERS-reporter approach has been more dominant over the last decade the merits of the label-free approach have begun to emerge with concomitant advancements in computational approaches. A review of research advances and developments in both methodologies is presented below. For general and exhaustive reviews of SERS applications in the biological context, also covering the SERS-reporter approach inside cells, the reader is referred to a few excellent recent publications.in cellulo environment. This is owed to spectral differences between the molecule's protonated and deprotonated forms . More recently, this 4-MBA pH sensing was multiplexed with simultaneous measurement of redox potential in live EAhy926 cells. The redox sensitive band at 1666 cm–1  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>P stretch) reports on the oxidation state of SERS reporter molecule N-[2- formamido]ethyl}disulfanyl)ethyl]-9,10-dioxo-9,10-dihydroanthracene-2-carboxamide (AQ), which was ratiometrically plotted against its redox insensitive 1606 cm–1 (CC stretch) vibrational mode. Cluster analysis then permitted quantitative analysis of redox potential dysregulation, a common indicator for the progressive pathology in neurodegeneration, cardiovascular diseases and cancer.The first and more comprehensively studied intracellular SERS technique is the SERS reporter approach, whereby NPs are functionalised with covalently bound, strongly Raman active organic molecules. Such molecules serve specific sensing purposes by binding to a target molecule. The generated SERS spectrum then reveals the defined signature of the label to facilitate indirect and highly sensitive detection of the target molecule for Raman sensing applications. A specific example of this concept is the use of 4-mercaptobenzoic acid (4-MBA) functionalised NPs as pH sensors, which are sensitive to changes between pH 6–8 within their  following subsequent advancement in synthesis to achieve ultrathin silica shells. Hollow AuNPs can also be protected by PEG capping, offering stabilisation and red shifting their SPR from 700 nm for detection by 785 and 1064 nm irradiation. Many SERS labels (reporter molecules) have been developed since the technique was established including enzymes, dyes, peptides and DNA which allow for multiplexed detection of numerous molecules using only a single laser wavelength. Multiplexing of SERS reporter functionalised nanoprobes allows for a much broader range of molecules to be simultaneously studied when compared with fluorescence microscopy, due to spectral resolutions of <2 nm which can enable access to between 10–100 unique optical signatures as provided by varying reporter molecules.The density of SERS labels on the surface of the plasmonic NP can be adjusted to experimental needs. For instance, the co-adsorption of other, undesired molecules as well as spectral interference can be avoided by complete coverage of the NP's surface with the label. Additionally, a (further) physical and chemical protection of the probes can be achieved by encapsulation with a silica layer as conceptually introduced by Mulvaney and co-workerset al. presented the first use of SERS reporter methodology to monitor the intracellular activity of specific enzymes, a tool which could possibly be implemented to detect dysfunction underlying many diseases. A recent example of this concept is the detection of carbon monoxide in normal human liver and HeLa cells. This sensing is based upon the sensitivity of SERS signal from 40 nm AuNP-bound palladacycle reporter ligands to carbonylation, induced by the presence of carbon monoxide, whereby the palladium constituent is removed from the ligand to form a carboxylic acid group as presented in  Inside cells, the presence of carbon monoxide releasing molecules (CORMs) was thus evidenced by the appearance of spectral bands at 1032 cm–1 (R–NH2 rocking), 1118 cm–1 (νCX stretch) and 1242 cm–1 (νCO stretch). The lowest detection limit of CORMs was found to be 0.5 μM in HeLa cells, attributed to the 1032 cm–1 vibrational peak. This finding offers potential to utilise palladacycle based SERS reporters as an analytical technique to develop a much required knowledge of pathophysiological events involved with carbon monoxide. These examples demonstrate the typical sensing-styled application of intracellular reporter SERS methodology as well as its high sensitivity.SERS reporter experiments benefit from the specificity of attaching a specific organic molecule to the NP and mapping its known SERS signature, suitable for various intracellular sensing applications. Stevenson in vivo in murine models with twice the sensitivity of two photon fluorescence techniques. However, SERS is of particular interest to oncology. Antibodies selective to proteins overexpressed in cancer cells have been used to target SERS reporter probes for diagnostic applications. Dual SERS-fluorescence diagnostics have been carried out using a fluorescently labelled aptamer for targeting protein tyrosine kinase, conjugated to Au–Ag nanorods also bound with Raman reporter molecule 4-aminothiophenol. SERS and fluorescent signals could be excited independently using different wavelengths of light for combined cancer cell recognition. Monoclonal antibodies have also been used to selectively target 40 nm AuNPs with squaraine dye reporter molecules to epidermal growth factor and p16/Ki-67 receptors to achieve selectivity to and recognition of cancer cells’ surface and nucleus. Therefore, SERS can be presented as a potential means of fast and accurate cancer diagnosis compared to time consuming immunocytochemistry methods.As a well understood technique with high specificity, SERS reporter methodologies have been developed into therapeutic applications such as the detection of inflammation as demonstrated  however this is mostly conducted at the cellular level. For instance, circulating tumour cells were detected in blood plasma by labelling of Ag–Au nanorods with a mixture of four SERS dyes, along with an ‘antibody rainbow cocktail’ of four breast cancer antibodies and a specific leukocyte CD-45 marker. This resulted in highly specific detection of a single cancer cell in a population of seven million blood cells when applied with multicolour and photothermal imaging. The presence of the SERS reporter dyes could be effectively imaged on the membrane of such tumour cells. Similarly, multicore SERS reporter labels functionalised with epithelial cell adhesion molecule antibodies have been shown to bind the membrane of living MCF-7 tumour cells without cellular uptake following just 25 ms exposure time. Potential for enumeration and sorting of circulating tumour cells is therefore very promising with microfluidic chips.Efforts have been made to combine the detection of cancer biomarkers with microfluidic technologies,et al. to not only achieve pH sensitive delivery of antitumour drug DOX into live cells, but also to monitor its release by SERS. Similar pH sensitive DOX release and SERS tracking has also been achieved by loading into multi-walled carbon nanotubes decorated with 4-MBA labelled Au and Ag core–shell NPs, verifying the drugs release inside lysosomes by imaging of its fluorescent signal with mapping SERS-determined cellular pH. Song et al. performed successful delivery and monitoring of DOX in live SKBR-3 cells using hollow, amphiphilic SERS-reporter vehicles. These particles consisted of 14 nm diameter AuNPs, coated with hydrophilic PEG, pH-sensitive hydrophobic PMMAVP grafts and the Raman reporter molecule BGLA (2-(4-(bis(4-(diethylamino)phenyl)(hydroxy)methyl)phenoxy)ethyl 5-pentanoate). The functionalised AuNPs were then self-assembled into hollow plasmonic vesicles tagged with HER2 antibodies for cancer cell targeting and loaded with DOX as shown in  Uncontrolled AuNP aggregation often causes inhomogeneity in SERS measurements (discussed in the next section on reporter-free SERS), yet has been manipulated in this study to yield a novel analysis technique. Although NP-based long term drug delivery has been employed for a significant length of time, the prospect of monitoring the release of the drug intracellularly using reporter SERS represents an exciting development across a range of therapies with suitable antibodies available. In addition to drug release and monitoring, AuNPs have been coated with bifunctional conducting polymer materials, acting as both Raman reporter molecule and a stable surface coating to offer purpose in photothermal therapy. These NPs are capable of highly efficient near-infrared photothermal transduction for cancer therapy whilst possessing good stability and biocompatibility both in vitro and in vivo in mouse models.Of great relevance to subcellular events is utilising SERS reporter NPs to achieve traceable intracellular drug delivery in therapeutics. A AgNP-loaded, graphene oxide based nanoplatform has been employed by Huang et al., who targeted 4-MBA-AgNP probes to the cell nucleus with HIV-1 protein derived TAT sequence modification of NPs to characterise their location and produce two dimensional SERS images of PC-3 human prostate cells. However, uncontrolled aggregation of spherical NPs not only causes locally heterogeneous enhancement of Raman signal by variable SERS hotspot generation, but also may prevent their internalisation or disrupt cell function due to the size of agglomerates. As a result, nanostars may be used for intracellular imaging and sensing. Those bound with nile blue, a dye used as a Raman reporter, and capped with bovine serum albumin have been utilised to map adenocarcinoma A549 and alveolar type II cells to identify constituents such as proteins, nucleic acids, lipids and carbohydrates as well as verifying that the SERS nanoprobes’ primary method of cellular internalisation was via endocytosis. Nanostars of 60–70 nm diameter, labelled with toluidine blue (TB) and encapsulated in a 30 nm coating of silica have also been employed for probing of the intracellular matrix. The performance of these particles was compared to spherical AuNPs with the same TB reporter and coating. Nanostars were shown by TEM to enter HeLa cells without any tendency to aggregate, albeit at a 3-fold reduction in internalisation. Upon irradiation with an 830 nm laser, SERS signal intensity from the nanostars was mapped across single cells at intensities greatly increased than that generated by spherical NPs, which barely exhibited a response. However, it must be noted when excited at 633 nm, a well-defined SERS signal could also be registered from the spherical nanoparticles, demonstrating the presence of the label within the silica shell. Therefore the intensity differences could be due to the excitation wavelength and plasmon resonance dependence of enhancements.Reporter SERS methodology can also be used to interrogate the intracellular distribution of various molecules by multiplexing the Raman signal of specific reporter molecules to produce SERS map images of single cells. The potential for this was demonstrated by Gregas et al. using nanotags functionalised with Raman reporters 4-mercaptopyridine, 5′5-dithiobis(2-nitrobenzoic acid) and 4-nitrobenzenethiol combined with multivariate analysis techniques. With data trends explained in all three dimensions simultaneously, multiple component detection was possible along with location of internalised nanotags was determined without the need for destructive TEM imaging. Such multi-marker approaches offer importance in delivering full characterisation of disease states or multiple cell organelle targeting while three dimensional analysis increases validity in modelling complex cellular structures.Three dimensional SERS elucidation of Chinese hamster ovary cell nuclei was achieved by McAughtrie –1) as a function of bound substituents.The probing of the intracellular chemical environment is very much a key application for SERS research given its high resolution and non-destructive nature. The ability to sensitively detect, map, monitor and quantify the localisation of both endogenous and extraneous molecules at the sub-cellular level would unlock a world of new information regarding the molecular changes undertaken by live cells during a range of cellular processes. The SERS reporter methods presented here offer promising steps towards visualisation and quantification of targeted molecules across individual cells. However, the effects of vast amounts and variety of biomolecules surrounding the nanoprobes go unaccounted for, limiting the amount of detailed information available as molecular events unfold. Despite the ability to multiplex data with numerous reporter molecules, this is a limitation of the technique when fully characterising cellular pathways. The approach may also lack spectral resolution in some applications where characteristic reporter bands (such as the phenyl ring modes of 4-MBA) only exhibit small wavenumber shifts (few cme.g. AuNPs). Molecules near or adsorbed onto an NP or into a NP's protein corona determine the measured SERS spectrum and allow for (bio)chemical sensing and characterisation, to track cellular processes such as cellular differentiation by DNA/RNA ratios, for instance. As mentioned earlier, single NPs can aggregate, giving rise to regions of intense enhancements called hotspots between the particles. This electromagnetic field enhancement between close-proximity SERS probes causes molecules located in those hotspots to provide a larger contribution to the measured SERS spectrum, albeit non-uniformly, as the aggregation and hence the generation of hotspots inside cells is largely uncontrolled. While this makes quantitative SERS measurements difficult, it allows for the qualitative but highly sensitive detection of the molecular environment inside cells.In contrast, in the reporter-free SERS approach the nanoparticles probe their direct vicinity and thus can potentially sense the large complexity of the chemical environment around them. In reality the affinity of molecules to the surface chemistry (charge) will determine the environment in the direct vicinity of plasmonic particles  can be particularly challenging especially in the more complex, eukaryotic  cells. Although examples of specific detection of molecules, wherein metabolites such as FAD and FMN have been identified in bacterial cells, the release and delivery of anticancer drug DOX was specifically tracked within live human oral squamous carcinoma (HSC-3) cells. AuNPs of 28 ± 3 nm diameter were functionalised with NLS and RGD peptides following (PEG)ylation, with the DOX bound to the Au surface by Ph-sensitive hydrazine linkage. Upon pH-dependent release of the drug within lysosomes, the intensity of its SERS band at 460 cm–1 was diminished with release over a 12 h period, with its inherent fluorescent signal no longer quenched by a close-proximity AuNP used to verify DOX localisation in lysosomes and resulting release into cytoplasm.Owing to the fact that this approach samples numerous constituents of the chemical environment around a NP, reporter-free SERS studies have been guided towards tracking and characterisation applications. In a similar technique outlined with SERS reporter methodology, Thus, controlling or even preventing this aggregation of NPs inside the cell in order to eliminate the incurred heterogeneity of acquired spectra from hotspots remains one of the key challenges to reporter-free SERS experiments inside cells. Novel structures, which show high plasmonic enhancements at the single nanoparticle level compared to single spherical nanoparticles, have been employed to overcome this hurdle, as are discussed later in this section.The Kneipp group carried out an early probing of cellular compartments by reporter-free SERS in living macrophages and endothelial cells. Differences in the SERS spectra obtained in different cell lines and over time, as well as the direct identification of physiologically relevant molecules, demonstrated that reporter-free SERS approaches are feasible for the characterisation of changing cellular environments and useful for intracellular applications. Additionally, the tendency of internalised AuNPs to uncontrollably aggregate results from the changing chemical environment experienced by the probes, with dimer and trimer formation  leading to increased enhancement of the Raman signal compared to individual particles.et al. a microfluidic chip was used for delivery and immobilisation of single cells. They used ordered continuous flow to expose Chinese hamster ovary (CHO) K1 cells to intracellular flux agonist ionomycin. Following mapping to reveal the location of the cell nucleus, cytoplasm and membrane, the approach was found capable of monitoring the chemical changes occurring within the cell during ionomycin-evoked Ca2+ flux response. This was achieved by observing the time profile of changing amide I (1643 cm–1) concentration, showing a similar trend to that of Ca2+ flux which is commonly used in biomedical research to stimulate the intracellular production of proteins such as interferons. This demonstrates the potential to utilise SERS for in situ monitoring of molecules to characterise protein expression dynamics at subcellular levels. The continuous flow methodology developed also permits more time effective experimentation when a series of SERS measurements are required, relative to batch preparation and analysis.Reporter-free SERS for mapping the intracellular environment has nevertheless been used in many applications. In a study conducted by Zhang  The SV-40 large T nuclear localisation signal peptide, containing a fluorescein (flu) tag at its C terminus for confirmation of attachment, was bound to 40 nm AuNPs via a cysteine linker and used to probe SH-SY5Y cells with 633 nm excitation, producing cellular maps at a resolution of 200 × 600 nm per pixel. Unambiguous characterisation of DC and undifferentiated cells (UDCs) was achieved through principal component analysis (PCA) across both the whole cell and the nuclear region. Furthermore, characterisation of cell differentiation was carried out by examination of nuclear regions, which undergo changes in ratios of molecular content as well as morphological and structural development. Reorganisation of chromatin plays an important role in the proliferation of non-dividing cells (DCs) which maintain a steady molecular formation relative to UDCs whose nuclear regions undergoes continuous changes in chromatin formation during mitosis. The acquired SERS data revealed a shift towards increased ratio of DNA/RNA in DCs relative to UDCs, as revealed by respective characteristic peak intensities. The number of protein peaks detected in DCs was also increased, indicating greater expression and variety of protein within the nuclear region. This characterisation of cellular differentiation exhibits not only the ability of reporter-free SERS to distinguish between cell phenotypes but also how the careful application of statistical analysis methods such as PCA are utilised to effectively reduce the complex data matrices to unambiguously achieve classification.Reporter-free SERS has also demonstrated the capability to differentiate between closely related cell phenotypes in progenitor and differentiated cell (DC) types. The novel method for creation of a reference state depicted in  whereby cells were subjected to a 72 h AuNP incubation pulse to ensure a wide distribution of the AuNPs within various types of vesicle and achieve high SERS signal. Cells analysed in this state were assigned as the sample group (n = 20). A reference state (n = 14) was generated by carrying out the same incubation phase followed by a 48 h depletion phase in which the extracellular fluid was removed and replaced with fresh, AuNP-free media to ensure that the nanoprobes were processed through the endolysosomal system and only localised within lysosomes. Background subtraction of the generated SERS map data with principal component  scores lower than 25% of the maximal PC1 value (reference state) with borderline case exclusion. Exemplarily, the back-projected pseudo colour map of the LD scores  and the classification was not only accurate but also demonstrated an advantage over single frequency SERS and simple LD score maps. Mean centring and PCA steps also address the issue of heterogeneous SERS enhancements by uncontrollable NP aggregation.The avenues by which the successful implementation of this tailored preparation and reference-based PCA-LDA based method address the shortcomings of reporter-free SERS experiments are multifaceted. Firstly, the technique not only allows for extraction of useful data from the complex and large original dataset, but also provides an improvement on existing analysis methods such as standard PCA-LDA  and the  NLS and RGD targeted Au nanocubes observed damage to cytosolic proteins containing sulphur and aromatic amino acids and changes to their secondary structure. The 502 cm–1 disulfide vibrational mode exhibited decreased intensity compared to that of the C–S 653 cm–1 mode during irradiation which reflected the photolytic cleavage of disulphide bonds. The UV-C exposure was also determined to completely arrest NP transport within living cells and induce apoptosis, hallmarked by intense vibrations at 1000 and 1584 cm–1. Real-time evaluation of defence mechanisms of cancerous cells towards UV exposure is a valuable application of reporter-free SERS in forwarding photothermal therapies.Intracellular and reporter-free SERS has also been utilised in elucidating the biomolecular dynamics of the stress response to UV-C (254 nm) irradiation in HSC-3 cells. The topology generated by surfactant mediated production from polyurethanes (PUs) is similar to that of a Faberge egg, with the hydrophobic core (used to cargo drugs) provided within the amphiphilic structure of PU globules. SERS monitoring of loaded pyrene was carried out, the release of which was shown to plateau out after 30 h. The lace nanoshells offer tighter synthetic control of diameter relative to thermosensitive polymer gold cages and liposome–gold nanocontainers also used for hydrophobic drug delivery. This aids avoidance of first capture by macrophages and renal clearance when carriers are too large or small respectively. The improved homogeneity of hotspots offers advantage in generating reproducible reporter-free SERS measurements over spherical NPs.As mentioned earlier, the uniformity of SERS signals is extremely significant for reporter-free SERS, in particular for quantitative analysis. Considerable efforts have been made into counteracting the uncontrollable aggregation of internalised AuNPs by adaptation and modification of the particle structure. The production of gold lace nanoshells not only generates high SERS enhancements in the 1–3 nm hotspots between branch like structures (3–5 nm) without aggregation, but also offers further application as a model for the delivery and monitoring of hydrophobic drugs. Like spherical NPs, the microchips were taken up by normal human dermal fibroblast (NHDF) cells with no suggestion of reduced viability from observation of mitotic events. A proof-of-concept detection experiment was then carried out using the common extraneous SERS molecule, rhodamine 6G (R6G). Following a 24 h incubation period, the intracellular location of the microchips and inhomogeneous distribution of R6G were confirmed in NHDF cells by confocal reflection and fluorescence microscopy (–1 R6G SERS peak (imaged in green).Similarly, the top-down construction of gold nanostructured microchips proposes an alternative means of removing the variability inherent to reporter-free SERS by aggregating spherical NPs. Chips with a uniform and reproducible nanodome gold pattern were generated by nanosphere lithography, depositing a gold nanofilm on a monolayer of polystyrene bead templates and thereafter released into solution.croscopy . The traThis successful detection of a well-established SERS-active compound using nanostructured microchips paves the way for detection of extraneous molecules within live cells with improved reproducibility due to relative homogeneity of hotspot enhancements. This study also partially addressed a previous difficulty in determination of the exact location of internalised AuNPs, given that voluntarily internalised NPs exist in membrane-bound transport vesicles from which they may or may not escape to be free in the cytosol. A lack of co-localisation of the microchips with fluorescent dyes LysoTracker Red DND-99 and Vybrant Dil (accumulating in acidic cellular components and lipophilic membranes respectively) suggested no localisation within vesicles of the endocytotic pathway, although further studies are required to verify exact location. Despite protein corona adsorption to the gold plated chips forming an extra barrier for molecules to cross to reach plasmonic hotspots, this novel and reporter-free approach takes positive steps towards homogenously enhanced, time-dependent and quantitative detection of intracellular molecules by SERS.et al. to simultaneously image the nucleus and plasma membrane of single HeLa cells in three dimensions, afforded by confocal SERS microscopy. Characterisation of the nucleus and tracking of time dependent changes in molecular constitution during induced apoptosis was achieved using NLS-functionalised, reporter-free AuNPs. Consequently, slow condensation and degradation of chromatin, nuclear rupture and denaturation and degradation of proteins were all identified. AuNP-bound 4-MBA, Crystal Violet and Cresyl Violet acetate were used as SERS reporters to monitor the localisation and detachment of folate and luteinizing hormone-releasing hormone receptors in real time. The SERS reporter portion of this investigation provided verification that specific endogenous molecules are being monitored, which is sometimes an ambiguity in purely reporter-free studies. Although a fairly laborious process, the combination of reporter and reporter-free SERS techniques displays great promise for in situ characterisation of complex intracellular processes, with application of suitable chemometric analysis.Recently, in a powerful combination, reporter-free SERS and SERS reporter nanoprobes were employed by Chen  Under appropriate laser powers, absence of signal deterioration effects such as photobleaching of samples allows for live cell investigations. Despite a requirement for standardisation of toxicity studies, the employed nanostructures – particularly those of gold – appear not to impinge upon the viability of live cells. Intracellular SERS studies are still in their infancy; however, this is a fast-paced field attracting high levels of interest with an ever-growing body of research. Advances in nanotechnology have led to an increasingly varied library of SERS active structures, tailored to addressing the difficulties of in cellulo experiments such as cellular entry, pH dependent aggregation and targeting of specific compartments.As a non-destructive, highly sensitive technique capable of sub-micron imaging resolutions, SERS holds immense potential for interrogation of the intracellular environment across biomedical research. Acquisition times can be optimised down to the sub-millisecond range, sufficient for even single molecule detection. The approach has matured to therapeutic application with multipurpose nanostructures which not only detect and monitor but deliver a cargo of hydrophobic drug molecules into intracellular compartments.It is clear that the choice of intracellular SERS experiment performed is very dependent on not only the studied system but also the data required. SERS reporter methodology benefits greatly from simplicity of data analysis, and holds advantage over fluorescence microscopy in increased spectral resolution to allow multiplexing of a larger number of unique optical signatures. The discussed studies offer reporter-based SERS as an effective tool for the imaging of molecules and proteins with known vibrational signatures, as well as relatively simple quantification of simple cellular processes. Combination of reporter-free and SERS reporter techniques yields comprehensive findings, tracking both the localisation of specific proteins as well as monitoring changes in the diverse chemical environment. Tailoring of sample preparation to create a sample set upon which to train multivariate analysis methods such as PCA-LDA prove equally capable with the potentially complex synthesis of multiple functionalised SERS reporter probes. In either case, it remains that the development of statistical methodologies and data analysis toolkits are central to potentiating the use of reporter-free SERS to deconvolute the molecular dynamics of intracellular structures, pathways and processes.Despite being less widely developed; reporter-free SERS is more suited as a powerful means of probing complex cellular processes due to the sheer wealth of information which can be extracted from a single measurement of the intracellular matrix about the vicinity of the NP. This however comes at the cost of throughput times, as careful processing and analysis of experimental data must be employed to extract desired information in order to accurately and unambiguously characterise processes at the molecular level. Such methodology was effectively achieved in the cases of endocytosis and apoptosis. Without information on whether candidates are entering cells, let alone localising to and interacting with target organelles or biomolecules, pharmacodynamics cannot be fully understood which contributes to the lack of in vivo efficacy responsible for the failure of a large portion of clinical trials. Microfluidics, both for nanoprobe synthesis and cellular interrogation will also play a major role in the development of tightly controlled, reproducible and ultimately higher throughput techniques. Along with early-stage diagnostics by SERS-reporter and reporter-free SERS methodologies, these applications should form central goals for future proof-of-concept, assay development and process optimisation studies in order to further develop and implement highly powerful intracellular SERS techniques for interrogation of the cellular environment.Future developments in intracellular SERS will be of particular interest to the pharmaceutical industry, for elucidating new drug targets in both healthy and pathogenic cells through cell-based assays. Additionally, there exists a requirement for determination of administered small molecules concentration and localisation at the sub cellular level, which no established technology can currently satisfy.


Emotional Design emerged as the effort to promote positive emotions , by providing inputs to digital technologies to modify or influence final outputs.This contribution explores opportunities provided by conceiving emotions as cognitive processes and active agents of interactions, in the field of emotional design.Affective computing studies . Studies on appraisal showed that an emotional episode emerges when one evaluates his/her own relationship with the surroundings Roseman, . This auappraisal—can be easily translated into initial guidelines to develop a cognitive science-informed emotional design.In our opinion, the scientific knowledge of discrete emotions based on their cognitive components—monitoring emotions), in order to tailor final outcomes. Future research in emotional design may explore how the continuous measurement of specific emotions can be exploited to influence ongoing interaction with common-use technology, for example modifying real-time easiness of use of devices or selecting digital content depending on the users' ongoing emotional responses.For instance, a field in which a partial discrete emotional approach was combined with affective is automotive technologies design (Ho and Spence, expand the kinds of emotions that designers can reproduce and promote. Insofar emotions are considered as discrete events emerging from a specific pattern of appraisal themes (Smith and Lazarus, emotional nuances a designer can detect and control. For instance, sadness' core appraisal concerns an irrevocable loss (Smith and Ellsworth, A lot have been done, but we argue that still more can be done relying on an appraisal-based discrete emotion design approach. Indeed, appraisal theories of emotion have a lot to offer emotional design Desmet, . Drawingcomplex emotional states which include several single discrete emotional sub-components. Indeed, intervening on aesthetic appeal of interfaces allows designers to promote a general positive feeling in users, that is what has been done by most current approaches. However, the scientific literature can provide indications to elicit even specific complex emotions simply basing on their pattern of appraisal. For instance, one is the emotion of awe or the deep feeling of wonder, astonishment and fear people experience when facing stimuli perceived as incredible and incommensurable (Keltner and Haidt, Such approach not only allows distinguishing different emotional nuances but it can also provide suggestions about reaching and promoting specific In the emotional design, another important aspect concerns that emotions are closely intertwined over a continuous stream within subjects' experience. The sub-components of emotional episodes influence each other and subsequent emotional responses. For example, sad people are more likely to attribute agency of subsequent stimuli to others and the external world, because sadness is an emotion experienced toward events one cannot control (Han et al., emotional profiles or pre-existing individual/contextual characteristics that can influence the effectiveness of emotion-based technological services. For example, smartphones can be designed to elicit reactions such as surprise (Desmet et al., In other words, emotions do not appear “out of nowhere” as the simple byproduct of a given stimulus and its appraisal. Instead, they are influenced by previous emotional states, or pre-existing individual traits, dispositions, and contextual factors (Verduyn and Brans, Finally, the advancement of common-use technology, combined with the knowledge available in cognitive science literature, could provide designers with extraordinary possibilities to fully exploit emotions' potential for user experience (see Figure ST conceived the ideas presented in the article and wrote the first draft. AC assisted in drafting the manuscript and contributed with important intellectual content. GLR edited the manuscript from a design perspective and created the image. GR supervised the whole process and contributed with important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous research has shown that fear-related stimuli presented in peripheral vision are preferentially processed over stimuli depicting other emotions. Furthermore, emotional content can influence dominance duration in binocular rivalry, with the period of dominance for an emotional image (e.g. a fearful face) being significantly longer than a neutral image . Experiment 1 of the current study combined these two ideas to investigate the role of emotion in binocular rivalry with face/house pairs viewed in the periphery. The results showed that faces were perceived as more dominant than houses, and fearful faces more so than neutral faces, even when viewed in the periphery. Experiment 2 extended this paradigm to present a rival pair in the periphery in each hemifield, with each eye either viewing the same stimulus in each location , or a different stimulus in each location (Diaz-Caneja condition). The results showed that the two pairs tended to rival in synchrony only in the traditional condition. Taken together, the results show that face dominance and emotion dominance in binocular rivalry persist in the periphery, and that interhemispheric interactions in binocular rivalry depend on an eye- as opposed to an object-based mechanism.


Studies of criteria-based assessment tools have demonstrated the feasibility of objectively evaluating eHealth interventions independent of empirical testing. However, current tools have not included some quality constructs associated with intervention outcome, such as persuasive design, behavior change, or therapeutic alliance. In addition, the generalizability of such tools has not been explicitly examined.The aim is to introduce the development and further analysis of the Enlight suite of measures, developed to incorporate the aforementioned concepts and address generalizability aspects.As a first step, a comprehensive systematic review was performed to identify relevant quality rating criteria in line with the PRISMA statement. These criteria were then categorized to create Enlight. The second step involved testing Enlight on 42 mobile apps and 42 Web-based programs (delivery mediums) targeting modifiable behaviors related to medical illness or mental health .A total of 476 criteria from 99 identified sources were used to build Enlight. The rating measures were divided into two sections: quality assessments and checklists. Quality assessments included usability, visual design, user engagement, content, therapeutic persuasiveness, therapeutic alliance, and general subjective evaluation. The checklists included credibility, privacy explanation, basic security, and evidence-based program ranking. The quality constructs exhibited excellent interrater reliability  and internal consistency , with similar results when separated into delivery mediums or clinical aims. Conditional probability analysis revealed that 100% of the programs that received a score of fair or above (≥3.0) in therapeutic persuasiveness or therapeutic alliance received the same range of scores in user engagement and content—a pattern that did not appear in the opposite direction. Preliminary concurrent validity analysis pointed to positive correlations of combined quality scores with selected variables. The combined score that did not include therapeutic persuasiveness and therapeutic alliance descriptively underperformed the other combined scores.This paper provides empirical evidence supporting the importance of persuasive design and therapeutic alliance within the context of a program’s evaluation. Reliability metrics and preliminary concurrent validity analysis indicate the potential of Enlight in examining eHealth programs regardless of delivery mediums and clinical aims. The wide distribution of personal digital devices has changed the potential to dramatically enhance public access to health interventions; tens of thousands of health, wellness, and medical apps are now available for download from online stores . From a Criteria-based rating scales have been developed to address this evaluation challenge. These scales are then used by trained raters to objectively examine and score the quality of eHealth intervention programs based on core concepts, each comprised of different criteria -11. The Persuasive design aims at understanding what influences people’s behavior and decision making, and then uses this information to design compelling user interactions ,16. InteNo previous quality rating scale has directly assessed the therapeutic alliance being nurtured by the eHealth intervention program. Overall, studies focusing on nontechnological interventions have shown that the therapeutic alliance is one of the most robust measures for predicting psychotherapy success eg, -23) and  and 23])Studies suggest that therapeutic alliances with eHealth intervention programs do exist and that such alliances may play a role in increasing the adherence to ,29 and eOverall, there is a need to develop standardized operational definitions to assess the quality of all aspects of eHealth intervention programs , includiAnother aspect that has yet to be fully examined is whether criteria-based rating tools may enable us to reliably rate eHealth intervention programs that are developed and designed to be used in different delivery mediums . Such reliable rating would enable examination of programs leveraging more than one delivery medium and comparison between the qualities of programs regardless of their delivery mediums. Recently, Gomez Quiñonez et al  demonstrThe gaps in the literature laid the foundation for the development of Enlight, a suite of criteria-based measurements aimed at enabling scholars to objectively rate eHealth interventions based on different quality concepts regardless of their delivery medium or clinical aims. Therefore, the aims of this study were to (1) identify relevant published criteria through a comprehensive systematic review that also addresses quality criteria related to persuasive design, behavior change, and therapeutic alliance/principles ; (2) develop Enlight, a suite of criteria-based quality measures related to separate aspects of eHealth programs; (3) establish the measures’ reliability and generalizability in evaluating different delivery mediums and clinical aims; and (d) examine the intercorrelations between different quality constructs and between them and preliminary validity measures.This study was completed in two parts, each reflecting different procedures used. The first part, “Enlight Development,” included a systematic search for quality criteria, the classification of these criteria into core domains and subcategories, and the creation of the different scales. The second part involved reliability testing and further analysis of the interrelationships between the quality constructs and correlations with preliminary concurrent validity measures. As emphasized, we examined the results with respect to different delivery mediums and clinical aims.The systematic review was carried out in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines   or mental health, and on programs delivered through mobile apps or websites . The systematic identification of relevant programs followed the PRISMA statement guidelines  . The inclusion criteria for mobile apps were (1) English language, (2) free of charge, and (3) from Android categories “Health & Fitness” and “Medical” following a careful examination of program type in different Android categories. To identify relevant Web-based programs, a systematic search was conducted on September 5, 2016, using a Google Search query per condition  paired with terms such as “free online” and “self-help.” For each condition, we looked into the organic results found within the first two pages because studies have indicated that a negligible portion of users go beyond the second page ,42. If aDifferent raters evaluated the quality (KF) and checklist (NM) sections of the programs, with the study’s leading author (AB) acting as the second independent rater for both sections. A total of 12 programs (the first three from the randomized lists for each of the four conditions) were used to pilot-test the scale. As part of this process, we also examined programs from other domains that were recommended by experts for their very high quality. This approach enabled coders to locate transcription errors and refine the coding scheme. One goal of the development was to achieve high interrater reliability at the construct level so that Enlight users could reliably present and compare construct scores between different programs. To achieve this goal, the refinement process followed the methods for developing benchmarks in thematic and projection test development within the psychology domain -46. DuriIn line with a previous examination in this field , a minimThis paper presents the analysis of all Enlight categories based on raters’ ratings, except for security checklist because security items are based on information retrieved from parties with access to products’ servers (and not on raters’ ratings). The interrater reliability for each of the quality assessment subscales was measured using an intraclass correlation coefficient (ICC)  utilizinP values based on the Benjamini-Hochberg correction )The results indicate that it is important to examine therapeutic persuasiveness and therapeutic alliance as part of the main quality constructs of eHealth intervention programs. Most importantly, a conditional probability analysis revealed that 100% of the eHealth intervention programs that received a score of fair or above in therapeutic persuasiveness or therapeutic alliance received the same range of scores in user engagement and content. For programs with fair or above scores in user engagement and content, only 33% to 64% of them received the same range of scores in therapeutic persuasiveness or therapeutic alliance. This means that, despite the strong, positive correlations found between the four aforementioned constructs, the relationships between them are more complicated: achieving a certain standard of scores in user engagement or content does not necessarily mean that this standard will be achieved in the therapeutic constructs and therefore justifies separate ratings of these constructs. Second, when examining the correlations between combined scores and variables that were expected to relate to either the program’s acceptability or its efficacy, the combined score that did not include therapeutic persuasiveness or alliance descriptively underperformed other combined scores, but the difference was small. Nevertheless, altogether these preliminary findings are congruent with findings from other studies, showing that persuasive design and behavior change principles are important factors in understanding the potential of eHealth intervention programs ,19,20. Tr: range .23-.41; all P ≤.02). At the construct level, user engagement, content, therapeutic persuasiveness, and therapeutic alliance demonstrated a pattern of positive correlations with these variables, although results were not always significant . These preliminary findings relate to Enlight’s concurrent validity, but they should be interpreted with caution because two of the selected variables only indirectly relate to program efficacy. Nevertheless, the program popularity score is related to a product’s acceptance and the evidence-based program score is directly related to the availability of sound research evidence on the product’s efficacy. These findings highlight the potential of this tool despite the need for further examination as discussed later.The analyses pointed to significant, positive correlations between the combined quality scores and credibility, evidence-based program, and program popularity scores .Several future directions for research and practice were identified. First, the predictive validity of Enlight could be examined by rating different programs for which metrics of acceptance and efficacy are accessible, and investigating whether and which quality scores predict engagement and efficacy. Second, it could be beneficial to develop a model of the various relationships between different constructs once many programs have been rated. Moreover, specifically examining therapeutic alliance and therapeutic persuasiveness could be helpful when trying to assess the additional need in human support to enhance adherence  or to prThis paper provides empirical findings that emphasize the importance of examining persuasive design and therapeutic alliance in the context of quality rating. It also demonstrates the applicability of objectively rating different eHealth interventions using one suite of measures, regardless of their delivery mediums or clinical aims, providing that raters are appropriately trained. The high reliability matrix and preliminary concurrent validity indicate the tool’s potential to examine eHealth programs and the multimodal relationships between different aspects of program quality. More research is needed to establish the tool’s validity for predicting the efficacy of eHealth programs.


This efficient method provides an excellent solution to postpone embryo transfer in natural cycles with adequate number and timing for main challenges of ART such as polycystic ovarian syndrome (PCOS), pre-implantation genetics diagnosis/screening (PGD/PGS), advanced female age, low responders, repeated implantation failure (RIF), multiple pregnancy, third-party reproduction, etc .The first baby was born from frozen-thawed embryos in Australia in 1984 and subsequently in USA in1986. Regarding limitations of cryopreservation techniques, its low efficiency and low post-thawing survival rate of embryos, less than 1% of all cycles were frozen-thawed embryo transfer (FET) at that time. It increased up to 30% in 2004 and this rate is growing not only due to its better success rate compared to fresh embryo transfer but also because of worldwide advocacy of elective single embryo transfer (eSET) over multiple embryo transfer. At recent, it is estimated that more than 50% of the children born following ART are from frozen-thawing cycles  babies in frozen cycles  in comparing freeze all protocols with fresh embryo transfer; however, most studies are not randomized and also there are four acceptable RCT works in different groups of infertile couples which are difficult to be compared. According to these results, benefits of freeze all strategy are likely for patients with good ovarian response and women with suboptimal/poor response had better results with fresh ET. Moreover, obstetric and perinatal outcomes regarding frozen ET are controversial .In addition, studies about cost and time concerns in freeze all policy do not seem to be valid since in some cases, the time spent and the cost per successful clinical pregnancy is less than that in fresh ET , 7.Since the health of ART born children during the whole length of life is important, considering above concerns, caution should be taken in using freeze all policy universally for all infertile couples. Therefore, more evidence is needed on the ramifications of freezing embryos and until then ART programs might adopt an individualized policy. Future studies may confirm the advantages of FET or refuse these findings and support fresh ET. Therefore, clinicians should carefully decide before adopting freeze all embryos as a beneficent policy for all patients.


This review provides a summary of recent insights into the role of the local white adipose tissue (WAT) in systemic sclerosis.Adipocytes located in an interfacial WAT area adjacent to fibrotic lesions have an intermediate phenotype and special properties implicated in fibrotic pathology in systemic sclerosis (SSc). The important role of these cells is recognized in different pathologies, such as wound healing, psoriasis, breast cancer, and prostate cancer. Additionally, both immature and mature adipocytes are involved in the appearance of fibroblast-like cells but exhibit different phenotypes and synthetic properties.Adipocytes from interfacial WAT adjacent to the fibrotic area in SSc are phenotypically different from bulk adipocytes and are involved in pathogenesis of SSc. Immature and mature adipocytes from this WAT layer differentiate into various types of fibroblast-like cells, making the local ratio of immature to mature adipocytes in interfacial WAT of particular importance in SSc pathogenesis. Systemic sclerosis (SSc) is an autoimmune rheumatic disease with an incompletely elucidated pathogenesis. Clinical manifestations of SSc include the development of fibrotic lesions in the skin and internal organs , 2. HoweIncreasing evidence suggests that white adipose tissue (WAT) is involved in the development of fibrotic lesions in SSc. Such involvement was first hypothesized and investigated at the systemic level. However, serum levels of different adipokines demonstrated no distinct correlations with SSc. Some authors reported no difference between patients with SSc and healthy controls , whereasRecently, the main interest in this field shifted to the role of adipocytes that are locally adjacent to the lesional tissue. Furthermore, we generally name this adipose tissue “interfacial WAT,” thus differentiating it from the bulk WAT at the same location. In this review, we discuss recent insights in this field.−/− mice demonstrate significant dermal fibrosis accompanied by a reduction of dermal WAT (dWAT) [The idea that interfacial WAT is involved in fibrosis is supported by multiple experimental findings. First, the appearance of dermal fibrosis is generally connected to some atrophy of the adjacent adipose tissue that generally occurs before the onset of fibrosis. Correlation of these two processes can be observed not only after bleomycin injections , 7•• butT (dWAT) .Second, immature and mature adipocytes have limited migration capacity in vivo ; thus, a−/− mice develop significantly milder bleomycin-induced pulmonary fibrosis [Third, several recent results demonstrated a correlation between microRNA (miR)-155 and the appearance of fibrotic lesions in SSc. On the one hand, miR-155 is involved in SSc pulmonary fibrosis •. Moreovfibrosis • and arefibrosis , 17, thuFourth, the involvement of adjacent adipose tissue was recently recognized in different pathologies in addition to SSc, including wound healing , psoriasAltogether, the adipocytes located near the interface with fibrotic lesions should have some special properties involved in fibrotic pathology. Moreover, significant differences should exist in the role of immature and mature adipocytes in this process.Fibroblast-like cells appear not only through differentiation of adipose-derived stem cells (ADSCs) but also from mature adipocytes that undergo phenotypic transformation. Replacement of the subcutaneous WAT (sWAT) by connective tissue in SSc was likely first described in , where iFurthermore, the thickness of the superficial WAT layer in SSc murine skin decreases with progression of dermal fibrosis , 25. IndThis step-by-step process prompted us to conclude that adipocytes located near the dermis/sWAT interface are involved in cutaneous fibrosis.Interfacial WAT in dermis is presented by dermal adipocytes, which are the fat cells located at the dermis/sWAT interface where they produce the layered structures in rodents and the typical cone-like structures around the pilosebaceous units that penetrate into the sWAT in humans •. ComparSkin histology reveals a correlation between the presence of such dermal cones and body areas that are typically susceptible to hypertrophic scarring , 29. OveIt was assumed that dermal adipocytes are phenotypically different from adipocytes located in the bulk of sWAT •. These Local thickness of the dWAT layer is mainly dependent on the number and volume of mature dermal adipocytes. This thickness varies both temporally and spatially, thus making the skin properties only quasi-constant . This thSynthetically active myofibroblasts can appear from resident fibroblasts through fibroblast-myofibroblast transition (FMT) , pericytAlthough atrophy of the adjacent adipose tissue generally precedes the appearance of dermal fibrosis, it is natural to assume that this effect is connected to the production of myofibroblasts from some cells present in adipose tissue. Recently, it was reported that myofibroblasts are produced from adiponectin-positive intradermal progenitors. This process was confirmed by adipocyte phenotype investigations ex vivo and is termed adipocyte-myofibroblast transition (AMT) ••. This Shortly afterwards, it was reported that resistin-like molecule α (RELMα/FIZZ1) triggers dedifferentiation of adipocytes and induces α-SMA expression in these cells , suggestTo the best of our knowledge, differentiation of myofibroblasts into adipogenic cells (MAT), which would be the reverse process to AMT, was only reported in vitro to date •. HoweveRecently, it was shown that β-catenin stabilization in fibroblasts from the lower dermis leads to a morphological modification that appears to be typical for the Wnt/β-catenin pathway: reduction of dWAT with simultaneous induction of dermal fibrosis , 50•. β-Given that dermal fibrosis is primarily connected to AMT, a correlation between dWAT properties and the severity of dermal fibrosis likely exists. Influence of the mouse strain on the severity of dermal fibrosis in a bleomycin-induced model was investigated in five common strains  in 6-week-old female mice . HistoloDifferent mouse strains have very different dWAT layers  and thusAdditional results obtained in  revealedThese findings demonstrate that dermal adipocytes should play an important role in dermal fibrosis and are potentially responsible for the appearance of myofibroblasts in lesional skin. Moreover, the results obtained in • supposeGiven that dermal and pulmonary fibroses have similar pathophysiologies and both involve the AMT, it can be assumed that lipofibroblasts play the role of dermal adipocytes in pulmonary fibrosis. These cells are located in the alveolar interstitium, involved in surfactant production , and can+ lung fibroblasts, suggesting that the activation of PPARγ may promote an adipo-epithelial phenotype typical of lipofibroblasts but not the myofibroblast phenotype [Lipofibroblasts have an intermediate adipo-epithelial phenotype  similar henotype .These two phenotypes do not exist separately from each other, and transdifferentiation of lipofibroblasts into myofibroblasts is documented in vitro . This trThus, transdifferentiation of lipofibroblasts into myofibroblasts in pulmonary fibrosis appears to be a very similar process to AMT originally described in dermal fibrosis. From this point of view, the “adipocyte”-myofibroblast transition is the universal process responsible for the appearance of different types of fibrosis in SSc. In addition, “adipocyte” should be understood as a chimeric cell with some intermediate and variable phenotypes.Breast cancer demonstrates a characteristic dense collagenous stroma. It is widely accepted that cancer-associated fibroblasts (CAFs) are involved in stroma production . It was Remarkably, mature adipocytes transform into ADF both in vitro and in vivo, and this transformation is morphologically connected with appearance of small adipocytes and elongated cells containing multiple small droplets. Such morphological features indicated that this intermediate phenotype can be connected with “beiging” of white adipocytes. Under proper conditions, human sWAT can indeed demonstrate the intensive beiging effect. Approximately 50% of omental adipocytes from patients with pheochromocytoma contain multilocular cells expressing UCP1 . TransdiIn contrast, under the influence of tumor-derived factors, ADSCs can differentiate into another CAF subpopulation , 74. TheAlthough this item was not investigated properly, it can be assumed that similar processes should occur in dermal fibrosis. The appearance of fibrosis should be dependent on the ratio of immature to mature adipocytes in interfacial dWAT, which can be modulated by different internal and external factors. In this context, it should be noted that bleomycin suppresses adipogenesis in ADSCs ; thus, tAll of these results lead to a general conclusion that adipose-derived progenitors and mature adipocytes should produce different types of fibroblast-like cells. Immature and mature adipocytes exhibit different levels of cytokine expression and drive stem cell signaling in breast cancer cells . MoreoveIt should be noted that this ratio is not constant and is increased in obesity. Moreover, high local expression of TNF-α suppresses adipocyte maturation, increasing the weight of immature adipocytes in the total adipogenic population , thus alThis important item can be illustrated using the recent results obtained for bone morphogenetic protein (BMP), which is the antagonist of TGF-β. BMP signaling is significantly decreased upon bleomycin exposure. In addition, endogenous activation of BMP reduces pulmonary fibrosis . In contDermal fibroblasts also demonstrate significant spatial phenotypic variations , which aIf immature adipocytes are mainly involved in the production of synthetic active myofibroblasts and the mature adipocytes transform into less synthetically active fibroblast-like cells, the local ratio of immature to mature adipocytes should be more carefully investigated in dermal fibrosis and other SSc pathologies. In this context, more severe bleomycin-induced dermal fibrosis is observed in male compared with female mice  and is pLocal WAT depots are involved in different pathological processes, including psoriasis, different types of cancer, wound healing, and fibrosis. There is increasing evidence that the adipocytes located near the surface of these depots should have phenotypical characteristics and physiological properties that differ from the classical adipogenic phenotype known for bulk sWAT adipocytes. Cells from the interfacial WAT layer adjacent to fibrotic lesions often demonstrate intermediate phenotypes, expressing the marker characteristic both for adipocytes and fibroblasts. This finding is not very surprising—given that these cells should simultaneously interact with classical fibroblasts and classical adipocytes in contrast to the bulk adipocytes.Although the WAT depots containing these cells differ in dermal and pulmonary fibrosis, the pathophysiological mechanisms of fibrosis in these two diseases appear to be similar. This finding points to the general involvement of adipocytes from interfacial WAT in SSc. Given that the subpopulations of adipocytes in such WAT depots are highly heterogeneous and that the immature and mature adipocytes utilize different pathways in the production of fibroblast-like cells and different migration properties, the structure and content of these adipogenic subpopulations in interfacial WAT depots in SSc should be investigated in more detail in the future experiments.