This review is dedicated to a comprehensive description of the latest achievements in the chemical functionalization routes and applications of carbon nanomaterials (CNMs), such as carbon nanotubes, graphene, and graphene nanoribbons. The review starts from the description of noncovalent and covalent exohedral modification approaches, as well as an endohedral functionalization method. After that, the methods to improve the functionalities of CNMs are highlighted. These methods include the functionalization for improving the hydrophilicity, biocompatibility, blood circulation time and tumor accumulation, and the cellular uptake and selectivity. The main part of this review includes the description of the applications of functionalized CNMs in bioimaging, drug delivery, and biosensors. Then, the toxicity studies of CNMs are highlighted. Finally, the further directions of the development of the field are presented. Carbon nanomaterials (CNMs), such as graphene, graphene oxide (GO), graphene quantum dots (GQDs), graphene nanoribbons (GNRs), and carbon nanotubes (CNTs), are of interest to the research community due to their outstanding chemical, optical, electrical, and mechanical properties ,2,3,4. TConventional chemotherapeutic drugs have often limited solubility in biologic media, low stability, and relatively high toxicity for normal cells and tissues and are missing a selective biodistribution. These shortcomings can be overcome if CNMs are used as targeted drug carrier and delivery systems . While sThe aim of this review is to comprehensively describe the latest achievements in the chemical functionalization routes and biological applications of CNTs, graphene, and its derivatives, such as GNRs, in biomedicine. The review has the following structure. It starts from the description of noncovalent and covalent exohedral modification approaches, as well as endohedral functionalization methods. After that, the improvement of the functionalities of CNMs is highlighted. These methods include the functionalization to improve hydrophilicity, biocompatibility, blood circulation time and tumor accumulation, and the cellular uptake and selectivity. Then, the applications of functionalized CNMs in bioimaging, drug delivery, and biosensors are reviewed. Finally, the toxicity studies of CNMs are highlighted.CNTs are hollow cylindrical molecules that consist of one or several rolled-up sheets of hexagonal networks (graphene) with at least one end typically capped with a hemisphere of buckyballs. The distinct property of CNTs is their high aspect ratio: their diameter is about one nanometer, while their length can exceed tens of micrometers. Unlike other nanomaterials, they can be metallic or semiconducting, depending on their chirality and add-atoms. This results in unique electronic, mechanical, and optical properties, ideal for a wide range of purposes . In addiractions . These pGraphene is a single layer of a honeycomb carbon lattice, the basic building block of all graphitic forms. Graphene and its oxidized derivatives (GO or reduced graphene oxide (rGO)) uniquely combine high electronic and thermal conductivities, mechanical strength, and impermeability to gases . Graphenications . In addiAnother recently invented and attractive biomaterial from the carbon family is GNRs, which is defined as a strip of graphene with a width less than 100 nm. GNRs are remarkable materials due to their attractive chemical, electrical, and optical properties . In compications .Each type of CNMs has advantages, disadvantages, and unique properties due to their distinctive characteristics. At the same time, CNTs, graphene, and GNRs share properties of an extremely small size and light weight, making them ideal candidates for nanoapplications. They all are resistant to ambient conditions especially to temperature changes from extreme cold to extreme heat and have unique mechanical properties. Moreover, they are not biodegradable. CNMs have large specific surface areas on which a wide range of chemicals can be attached. Therefore, CNMs can be functionalized to enhance their physicochemical properties and to deliver molecules and even large particles. In addition, CNMs are highly conducting in nature, provided they have a particular structure. In comparison to graphene, CNT and GNRs have optical properties, for example bright photoluminescence (PL), which are highly related to their geometrical structure.At the moment, the process is relatively expensive to produce CNMs, and in most cases, only small quantities of the material can be produced. Despite the advantages of using CNMs, it would be difficult to replace the older well-established technology with a new one. The lack of solubility in aqueous media, as well as their toxicity caused by the hydrophobic surface make CNMs difficult for some specific applications. An additional factor that will affect CNMs’ applications is the ability to form large colloidal aggregates without functionalization. CNMs cannot survive from oxygen along with heat. Furthermore, their properties are highly dependent on their size, which in some cases is difficult to control. Scaling up CNMs to useful sizes has not produced materials with the same properties.Chemical functionalization of CNMs is performed by two methods: exohedral and endohedral functionalization. There are two techniques for the surface functionalization of CNMs: noncovalent and covalent functionalization. In the next sections, we provide an overview of the chemical functionalization approaches of CNMs .Noncovalent functionalization relies on other, generally weaker, but also reversible interactions. These can be hydrophobic interactions,  reviews ,26.Covalent functionalization is the covalent bonding of desired functional groups on the surface of CNMs. The stable bonds enable a better process control as compared to noncovalent functionalization. The covalent bond distorts the conjugated Refluxing and sonication in oxygen-containing mineral acids and applying ozone or hydrogen peroxide are established methods for the oxidative treatment of CNMs. The oxygen-containing groups that are created by the initial oxidative treatment become the anchor sites for esterification or amidation reactions. These are widely employed for the conjugation of water-soluble organic molecules, hydrophilic polymers such as polyethylene glycol, nucleic acids, or peptides. These steps yield a wide variety of multifunctional CNMs. Another common approach is cycloaddition reactions by 1,3-dipolar nitrene and carbene. Such functional groups can be substituted with amino acids, peptides, and fluorescent molecules. The covalent functionalization via radical addition reactions is conducted through aryl diazonium coupling. For more information, reader can refer to the reviews ,27.If the chemical functionalization of CNTs is performed endohedrally, the functional molecules are encapsulated inside the channels of nanotubes ,39,40,41The technique to achieve an endohedral functionalization or simply filling of CNTs has to be chosen according to the chemical ad physical stability of the respective filler molecules or substance ,48,49,50 of CNTs ,53,63,64In the literature, there are reports on methods to improve the functionality of CNMs in biomedicine. These methods can be classified into four main directions, such as functionalization for (i) improving hydrophilicity, (ii) biocompatibility, (iii) the blood circulation time and tumor accumulation, and (iv) the cellular uptake and selectivity  65]..65].CNMs should be soluble in aqueous solutions and preserve their physicochemical properties in the medium used for their administration and in biological fluids. To achieve hydrophilicity, CNMs such as GO are usually covalently functionalized with hydrophilic polymers containing amine groups through the formation of amide bonds.Several studies ,67 showeThe biocompatibility of CNMs is influenced by the intrinsic properties of the materials, such as the surface chemistry, size, stability, purity, as well as by factors related to assay conditions, such as culture conditions, presence of serum, and cell line ,77. The Therefore, surface functionalization of graphene using biocompatible polymers to make it more water soluble and to improve its dispersibility are commonly used. In past works ,79, GO wriod. In , the in CNMs are not suited to overcome the biological barriers required to achieve a suitable tumor accumulation. Upon introduction to the blood stream, CNMs adsorb proteins and suffer internalization by macrophages . The funAfter reaching the tumor site, CNMs must be internalized by cancer cells. The functionalization of CNMs can improve the cellular uptake and selectivity. Methods of the efficient functionalization of CNMs have been developed. In several studies ,93, branChemically functionalized CNMs can be used for bioimaging and in vivo monitoring of processes in living cells, tissues, or even whole bodies. Such tools are relevant for research and diagnostics alike. Two basic requirements have to be met for bioimaging. There needs to be a fast and sensitive method for detection, and the contrast agents or spectroscopic markers must meet all the requirements, i.e., biodegradability, biocompatibility, high specificity, sensitivity, and general applicability ,102,103.Magnetic resonance imaging (MRI) requires magnetic atoms in a contrast agent such as gadolinium (III) salts, iron oxide, or nitroxide radicals . Functio2O3  and radi2O3 [2  and 125I2 [125I−  have beed SWCNTs , neutroncapsules , and astd SWCNTs  have beed SWCNTs  and antind LuCl3 . SWCNTs ticulum) .In , a wholem@MWCNTs . The accm@MWCNTs a. The blm@MWCNTs b. The exectively c. Neglignd liver d.Nanomaterials have been extensively used for drug delivery applications ,118,119.The development of drug delivery systems based on endohedrally and exohedrally functionalized CNMs is being actively researched. The research is focused on further developing and optimizing the methods of the chemical functionalization of CNMs. Increasing the degree of loaded therapeutic drugs and reducing the amount of nonloaded drugs are also important. There are methods to control the release of drugs, but better and more applicable methods are needed to achieve more efficient therapies. The solubility, biocompatibility, blood circulation time, biodegradability, and targeting of functionalized CNMs have to be improved, while their toxicity to normal cells has to be minimized. For these improvements, the underlying chemical and physical properties of the chemically functionalized CNMs need to be studied in detail. The therapeutic options can be further expanded by combining different agents, i.e., the codelivery of two and more drugs in functionalized CNMs. The development of a combined delivery of both biomedical contrast agents and drugs will in combination with a controllable release mechanism allow for an early diagnosis and local treatment of diseased tissues.Nanomaterials have also been actively studied as biosensing agents ,129,130.Glucose detection is one of the important applications of biosensors due to the need to control diabetes. Several promising CNT-glucose biosensors based on glucose oxidase were developed in 2004 ,133. VilIt was found that single-stranded DNA absorbs strongly on CNTs in comparison to double-stranded DNA. This property can be used to build CNT–DNA biocomplexes for biosensing technologies to target various molecules . The DNAMWNTs were also used in biosensors based on microsomal cytochrome P450 to detect drugs used in the treatment of breast cancer . D±-galalactin-3 . Folic-alactin-3 .Overall, CNT-based biosensors have many advantages over commercially available sensors. Among them are the high sensitivity, due to their high surface-to-volume ratio and hollow tubular structure, fast response time, due to the fast electron transfer reactions, long lifespan, and high stability.In addition to CNTs, graphene and its derivatives are extensively explored for the improvement of the existing contrast agents, as well as developing completely new probes and agents in biomedical imaging. Different techniques are used to monitor the processes, not only in living cells, but also in tissues and the whole body .As mentioned earlier, fictionalization is a key task of any type of imaging as it prevents the agglomeration of graphene in physiological buffers, which reduces the contrast and spatial resolution, in addition to increasing the biocompatibility. It is important to note that graphene itself is hydrophobic in nature , which mUnlike gapless graphene, GO shows a broad-band photoluminescence (PL) in the visible and nearinfrared (NIR) range excited by ultraviolet (UV). The usage of this intrinsic PL of nanographene oxide for NIR imaging of lymphoma cells was reported in . GO has The strong D and G Raman modes commonly observed in graphene can be further enhanced by noble metal nanoparticles’ deposition. The gold and silver nanoparticles on the graphene surface result in surface-enhanced Raman scattering (SERS). GO decorated by gold nanoparticles used for Raman imaging of HeLa229 cells showed a stronger and up to 192 better contrasted images than for GO . The silThe two-photon fluorescence of graphene-based materials results in creating highly efficient contrast agents for deep-sited organs due to the infrared two-photon excitation located in the NIR and middle-infrared (MIR) ranges in biological transparency windows. As an example, the growth of a whole zebrafish embryo treated by GO was dynamically imaged by using two-photon fluorescence . The nitTo overcome the penetration depth limit and quenching effect, typical for optical methods, radionuclide-based imaging methods are commonly used. GO edges and defects are used to attach a number of labels such as behavior ,166,167.ribed in . The tar kidneys .In contrast to the above-described methods, MRI is a noninvasive method with a high spatial resolution. GO is a diamagnetic material and cannot be considered as a contrast agent for magnetic resonance imaging. Only the manipulation with defects and oxygen groups of GO allow finding magnetism in this structure. A positive T1 MRI GO contrast agent (GO–DTPA–Gd/DOX) based on GO–gadolinium (Gd) complexes offered a dual-modality: MRI/fluorescence imaging and drug delivery . In part–0 cells . The conFinally, to overcome the problem with a penetration depth, photoacoustic imaging (PIA) based on graphene can be used. Graphene-based nanomaterials have been widely investigated due to the high NIR absorption and conversion to acoustic waves. The rGO and iron oxide nanoparticles were used as a contrast agent in photoacoustic tomography . The indThe areas of graphene in GO contain free al drugs . In addial drugs . In addial drugs . Most ofThe drug delivery capability of GO was reported for the first time by Liu and coworkers . In thisGraphene–carbon nanotube–iron oxide binds the anticancer drug 5-FU, leading to a high loading capability and pH-dependent drug release . A multir uptake . Anotherr uptake . Anotherr uptake .The efficient treatment of hepatitis in mice was performed by graphene QDs via the preferential accumulation of the liver and the reduction of Con A-mediated liver damage . PrimidoImportantly, GQDs show an inhibitory activity related to drug-resistant viruses and bacteria . GO alsoGraphene was shown as an effective intracellular and protein delivery agent ,193. TheGraphene and its derivatives have been employed in the design of different biosensors of various types because of their large surface area, bright fluorescence, high electrical conductivity, and high electron transfer rate. Graphene has been extensively explored for glucose sensors . Severalomplexes .Graphene was also employed as a sensitive substrate for fluorescence detection systems . It was -amyloid . A unive-amyloid , glucose-amyloid , and pes-amyloid . As a ze-amyloid ,222. The-amyloid . Further-amyloid ,225. The-amyloid .Unlike graphene, the lateral quantum confinement imposed by their finite width opens a sizable electronic band gap, which makes GNRs and its derivatives highly suitable for optical imaging . The firractions . The GNRractions .With diffraction-limited fluorescence microscopy and super-resolution microscopy imaging techniques, the structure of single-dye-functionalized GNRs coated on transparent and opaque insulating substrates from dilute solution can be resolved. oGNRs can also be reduced to rGNRs, a modification that also improves the NIR absorption and photothermal capacity, but reduces the solubility . Gd3+-iomography .In comparison to the rest of the carbon-based materials, the usage of GNRs arises from the combination of the abundant oxygen-containing functionalities and the large loading surface area. oGNRs and rGNRs are used in cancer therapy and can be functionalized by the same procedures described for GO. Therefore, GNRs have been proposed as a unique drug delivery agent for tumor therapy. The phospholipid–polymer-conjugate-functionalized oGNRs, oGNR–PEG–DSPE, can be considered a good vessel for delivering lucanthone (Luc) to glioblastoma multiform (GBM) tumors . Higher ma cells . DSPE–PEma cells .Polyethylenimine-grafted graphene nanoribbons (PEI-g–GNRs) were proposed as an effective gene vector drug carrier with the targeting of HeLa cells. Moreover, a novel oxidized graphene nanoribbon-based platform (oGNR) for gene delivery of double-stranded DNA into mammalian cells was performed. It exhibited efficient DNA loading of small double-stranded (ds) DNA fragments . Some stNonoxidized GNRs may be more appropriate for use as biosensors in which the higher surface area is a key factor for assembling polymers and DNA . In addiNanomaterials have been used in numerous biomedical applications. However, considerable attention needs to be paid to the human and environmental risks of these materials. For this reason, it is important to evaluate the level and degree of toxicity, biocompatibility, and biodegradation of nanomaterials ,252,253.In comparison to the rest of the carbon-based nanomaterials, CNTs containing catalyst metal ions that are incorporated inside of the CNTs are toxic to cells . These mThe shape, surface chemistry, purity, morphology, surface charge, functionalization, dispersion, and number of layers play important roles in the toxicity of graphene . Traditiacturing . The toxacturing . In compg [μg/mL .Similar to CNTs, the toxicity of graphene highly depends on the functionalization. It has been shown that carboxyl functionalization can significantly decrease graphene’s toxicity . HoweverIn this review, we comprehensively described the latest achievements in the chemical functionalization routes and applications of CNTs, graphene, and GNRs. We discussed noncovalent and covalent exohedral modification approaches, as well as endohedral functionalization methods. The methods to improve the functionalities of CNMs were highlighted. In addition, the applications of functionalized CNMs in bioimaging, drug delivery, and biosensors were extensively reviewed. The study of the biocompatibility of GNMs is still limited. Therefore, we summarized the toxicity studies of CNMs. Overall, this review tracked several challenges, for example the appropriate functionalization and biocompatibility, that need to be resolved to improve the bio-applications and to bring them from the research laboratory to the clinic. We believe that our review will also help to guide future research.The development of methods of the surface functionalization of CNMs to improve the solubility, biocompatibility, biodegradability, and targeting of functionalized CNMs and reduce their toxicity;The development of methods of the loading of CNMs with therapeutic drugs to increase the degree of loading and reduce the amount of nonloaded drugs;The comparison of the efficiency of the methods of the external and internal loading of CNTs with therapeutic drugs;The revealing of the correlation among the structural parameters of CNMs , surface functionalization, and their accumulation in tissues and cells;The development of the methods of controlling the release of drugs from loaded CNMs ;The development of the methods of the loading of CNMs with contrast agents for bioimaging to increase the degree of loading and reduce the amount of nonloaded contrast agents;The controllable modification of the physicochemical properties of CNMs loaded with contrast agents to improve the sensitivity of imaging and detection, to provide a high spatial resolution and imaging of deeper tissues;The development of the methods of the combined delivery of both biomedical contrast agents and therapeutic drugs using CNMs.The further directions for the development of the field include:


The Washington Group questions (WGQ) on functioning have been widely promoted as the go-to tool for disability data collection. Designed for use by government, the WGQ have been adopted by non-government organizations (NGOs) for use in programming. However, little is known about how the WGQs are being used by NGOs or how use may be contributing to disability inclusion. Method: This paper describes exploratory research on the use of the WGQ in NGO programming. An online survey provided an overview of adoption followed by semi-structured interviews from a purposive sample to explore data collection, analysis, and use. Results: Thematic analysis showed limited inclusion outcomes directly attributable to use of the WGQ, adoption driven by individual champions rather than systematically across organizations, and challenges in data collection resulting in a wide range of prevalence rates. What information the WGQ can realistically contribute to programs was also overestimated. However, the process of using the WGQ was raising awareness on disability inclusion within program teams and communities. Conclusion: Acknowledging differences in emerging use by NGOs beyond the WGQ’s intended purpose, alongside promoting a flexible and staged approach to adoption and use in programming, may improve utility and disability inclusion outcomes over time. The Washington Group on Disability Statistics is a United Nations (UN) City Group established in 2001 to improve disability data collection and comparisons between countries [The context for this study is the increasing use of the WGQ beyond government and their original intended purpose. Non-government organizations (NGOs) are using and recommending use of the questions in development and humanitarian programming ,4,5 and The WGQ use activity limitations as an indicator of functioning to identify people at risk of disability . The queThe foundational question set is the Short Set of six questions. There are also Enhanced and Extended Sets and a Child Functioning Module (CFM) designed with the UN Children’s Fund (UNICEF). A Labour Force Disability Survey Module has been developed with the International Labour Organization (ILO) and an Inclusive Education Module is in development with UNICEF. The question sets do not use the term ‘disability’ to avoid issues arising from differing understandings of disability and underreporting due to stigma . The WGQRecent studies of use in government surveys and related programs have mostly focused on issues relating to disability prevalence. A study of use in population surveys identified prevalence figures from approximately 3% to 20% . ContribIn comparison, and despite the availability of NGO guidelines and standards, evidence on use of the WGQ in non-government programming remains largely anecdotal. Similar to the above, this exploratory study finds use of the WGQ by NGOs is resulting in varied prevalence figures. Additionally, we identify how practical issues in data collection by NGOs contribute to these variations. We note the need for more realistic consideration of what the questions may contribute to NGO programming and the need for a more flexible approach to use aligned with better identifying people with disability for inclusion in programs. While we found limited evidence of improved disability inclusion outcomes arising from use of the questions, we found processes of adoption were raising awareness and improving understandings of disability.This research was conducted in partnership with CBM Australia under the Australian Government funded Partnership for Provision of Disability Technical Advice and Services through the Department of Foreign Affairs and Trade (DFAT) (Grant agreement number 74096). Ethics approval was from the Melbourne School of Population and Global Health Human Ethics Advisory Group (Number 1852115.1).The study included two components. The first was an online survey to provide an overview of use of the WGQ and to identify semi-structured interview participants. All study participants were adult (18 years of age or older) professionals working in international development and/or humanitarian action. Inclusion criteria was having organizational experience of using any of the question sets in design, implementation, monitoring, or other program activity.Online survey respondents were identified via key informants from the Nossal Institute, CBM Australia, and the Australian Disability and Development Consortium (ADDC). Recruitment emails included the option to pass on the invitation to relevant partners or colleagues. Three government and three research institutions who did not use the questions in direct programming activities were excluded from the analysis of online survey data. Survey participants could opt in for participation in the follow-up interviews. Associates who had used the questions in work related to the Nossal Institute were excluded. Where there were more than one respondent from an organization, one participant was selected in consultation with the organization concerned. Participants from 11 organizations successfully completed interviews between November and December 2018. The research process, including participant numbers and key topics of enquiry, is summarized in Interviews were conducted by telephone or non-video internet call for consistency and were recorded with the participant’s consent. One researcher led each interview using an interview guide and another took notes. Interviews were completed within 60 min with the exception of one slightly longer interview. Interview notes were checked against recordings and were shared with the respondent for review and approval. The final respondent-checked interview notes were imported into NVivo for coding and thematic analysis. A sample of interview notes were independently coded by two researchers. The researchers then reviewed the other’s sample notes and the code list was refined. This list was trialed by a third researcher on a further sample of interviews resulting in a final coding list. This list was used by the researchers to identify themes through an iterative process of comparison and discussion. Preliminary findings were shared in a workshop with project partners and experts, including Associates excluded from the semi-structured interviews. Feedback resulted in no major changes to the identified themes; however, the importance of distinguishing uses of the questions was highlighted. Use of the questions for data disaggregation is emphasized in guidance for programs but this was not the main use identified.As noted, this research was exploratory in nature and has limitations. We focused solely on organizations that had used the WGQ. We only interviewed one representative from each organization to avoid bias (anticipated from the online survey) from reporting on the use of a one question set across similar programs by one organization. All respondents had a prior interest or organizational focus on disability inclusion. We did not include organizations that had not used the questions and cannot speak to wider barriers to adoption. Our interview findings, which are the focus of this paper, are based on a small sample with experiences of using the questions concentrated in Southeast Asia and the Pacific. Caution should be exercised in making generalizations from the findings from this study. We are conscious of the different operational realities of humanitarian and development programming; however, we consider the findings we present to be of relevance to both.Organizational characteristics and use of the questions reported by online survey respondents are summarized in n = 12) compared to difficult to use (n = 13). From these, two said the questions were very easy to use and one said very difficult. The majority of respondents (n = 21) felt using the questions helped with their work with most saying the questions helped a lot (n = 13) and the remainder saying it helped a little (n = 8). Only one respondent reported the questions as not being helpful with three respondents unsure. How the questions were used and were contributing to inclusion outcomes was explored in the semi-structured interviews.An approximately equal number of online respondents reported the questions as easy to use (n = 5), Southeast Asia (n = 5), South Asia (n = 1), Central Asia (n = 1), and Africa (n = 2). Findings are presented below based on the themes summarized in Interview participants were from eight international NGOs, one national NGO, and two organizations of people with disability (OPDs). These organizations reported on work in the Pacific . Adoption of the questions was internally driven by motivated individuals in country offices or at headquarters.Reasons for using the questions included consistency with the organization’s inclusion strategy or rights-based approach, growing interest in disability inclusion in the wider sector they work in, and avoiding reliance on unreliable or dated local or village government data. Almost all organizations reported only using the questions in individual projects rather than broadly across their programming. Few reported the targeted use of the questions to answer specific questions or address particular inclusion issues in projects. The simplicity of the questions was noted as positively influencing uptake and foundational Washington Group concerns, such as the need for accurate, standardized, and comparable data, were echoed by respondents.Interview participants used the questions in a range of sectors, including education; health; nutrition; water, sanitation, and hygiene (WASH); climate change; disaster risk reduction (DRR); and humanitarian response. Project examples included improving mother and child nutrition in the Pacific, increasing access to education in Central Asia, and emergency shelter assessments during humanitarian response in Southeast Asia. The questions were used in household surveys, screening tools, participant lists, and post-disaster needs assessments.The majority of participants had accessed guidance from the Washington Group’s website. Published guidance for NGOs also draw on the Group’s guidelines. One organization had received training from Washington Group members and another from UNICEF on the CFM. Participants noted that individual questions, according to guidance, should not be changed, but most recognized the questions were adjusted in practice and that this was hard to monitor. Some changes were deliberate and in line with Washington Group guidance, such as changing the example for distance walked in the CFM from the length of a football pitch to between two electricity pylons. Another noted they did not mention hearing aids in the question on hearing as they were not available in their working area. Participants were aware that disability should not be mentioned when administering the questions, with one disguising their organization’s name and others introducing the survey as health, DRR, or WASH surveys. However, it was recognized that data collectors commonly mentioned disability out of habit.NGO-focused guidance recommends the inclusion of data collectors with disability, but only one organization reported this as standard practice. For others, including people with disability in data collection was opportunistic and dependent on the availability of a known OPD. The limited availability of OPDs in working areas was considered challenging. Including data collectors with disability was reported as time intensive, and one participant noted necessary organizational systems may not be in place. For example, no budget allocation existed for reasonable accommodation, including the covering of travel costs or individual support. Organizations that had included people with disability in data collection reported positive experiences. One noted that this was critical to identifying people with disability in communities and including people with disability in trainings on use of the questions was more effective. Another noted secondary benefits with OPD participation in data collection leading to new working relationships between the OPD and local government.The Washington Group recommends the questions be asked to all members of a household with the CFM asked to the mother or primary care giver. Not all participants had oversight of who answered the questions. It was assumed, albeit not condoned, that data collectors asked whoever was at home. Some noted asking the questions at the household level inevitably led to answers being provided by the head of household. One organization followed up with any individual with disability identified by the head of household. Another noted that, if asked, household members would only ‘bring out’ a person who had been previously identified as having a disability. One organization changed their approach to save time and now only asked the person ‘suspected’ to have a disability. Another noted their approach varied depending on available resources.Most participants used the WGQ alongside other questions. Only one organization, working in WASH, reported using the questions in a large household survey with multiple other questions. One organization used the Short Set as a stand-alone tool with no additional questions. In general, few questions were asked alongside the WGQ. Questions on gender were usually included with some respondents including questions on use of assistive products, transportation needs, or allergies and other health issues. One organization asked whether the person identified as being a person with a disability after the WGQ had been asked.Some modifications to the Short Set were reported with two organizations adding a question on hand function. A WASH actor added a question on turning on taps. Another added a hand function question for use in livelihoods interventions that was adopted from a livelihood survey form used by the national statistics office. One of these organizations recognized that the question on self-care addresses hand function; however, they considered this insufficient as people adapted to difficulties in dressing and washing and underreported difficulties. No organizations reported using less than the full six questions in the Short Set.The availability of translations was reported as an issue. Participants noted guidance on translation would be helpful and were not aware of the translation protocol available from the Washington Group . Few orgTranslation challenges included differentiating between ‘remembering’ and ‘concentrating’ in question six and between the ‘some difficulty’ and ‘a lot of difficulty’ response categories. Question five on self-care was considered hard to translate by several participants. The issue of ‘cultural overlay’ and what is ‘normal behavior’ for children was noted as an issue in the CFM. As a result of these issues, data collectors often explain and translate on-the-fly. This could be to add context, be due to working from written English survey forms, or the need to translate from a national language into a local language or dialect. Training data collectors to anticipate difficulties and to provide standardized explanations, such as drawing on optional questions in the Extended Set, was not usual practice.Analysis of data was descriptive and based on simple counts of the functioning difficulty recorded. Some participants disaggregated the disability data collected by gender and age. While simple counts and determining percentages across domains did not present issues, respondents felt they lacked knowledge on, or clear guidance for, more detailed comparative analysis. Participants found guidance on analysis from the Washington Group website to be unclear . OverallParticipants were aware the ‘a lot of difficulty’ cut off point is recommended for use in censuses and national surveys to identify people with disability. Several participants noted including ‘some difficulty’ responses was important for increasing program participation. Another reflected on the subjective nature of responses and reported some senior OPD members with disability would answer ‘some difficulty’ when asked. One participant said including ‘some difficulty’ responses had been an ‘eye opener’, as it identified people they had not considered before. One organization counted people who answered some difficulty in at least two domains as a person with disability. Several organizations considered it a requirement to use the ‘a lot of difficulty’ cut off point; however, one reflected that this probably meant they were missing people with disabilities.The main reported use of data was to identify people with disability for inclusion in program activities; however, this was not without issues. One participant noted the data confirmed what they already knew; that is, that there were people with disability in their working area. While one noted consistently higher figures than from local government data, another questioned the high prevalence figures they were finding. Another noted they were finding 1% to 2% prevalence when using the CFM and were concerned this did not reflect the reality on the ground. Inadequate training of enumerators was noted as a contributing factor. One participant noted prevalence data from work in different countries ranged from 1%, attributed to data collectors persisting in first asking who in the household had a disability, to what they considered a more realistic 15%.The questions were reported as being used to measure the participation of people with disability in program activities. This was often limited to measuring the attendance of people with disability in an activity, such as a training or workshop. No examples of assessing the extent of participation were provided, including what proportion of people with disability from the overall community took part in activities. Several respondents used the questions to inform accessibility measures for workshops and program activities. Examples included conducting preparatory activities with participants with disability, ensuring physical accessibility of venues, and preparing information in alternative formats. One respondent reflected that the need for improved accessibility exists, and is independent from, whether this data is collected or not.Concrete examples of how Washington Group data was contributing to improved disability inclusion outcomes were limited. One participant observed that despite using the questions, evidence of increased participation of people with disability in their programs remained anecdotal. One noted it was too early to tell if using the questions was increasing participation of people with a disability or not. Two organizations used the questions as a screening tool for more in-depth follow up. One used the Short Set to rapidly screen households for people with disability in new working areas. The other used the CFM to screen children with disability in schools prior to individual assessments. This was reported as aiding the development of teacher training materials and improving classroom practice. Washington Group data was also used for advocacy, including to deliver ‘stronger’ messaging to government. As discussed below, the most obvious impact was how the process of using the questions was contributing to change.The online survey showed the WGQ are being used in a range of programming contexts; however, the interviews suggested applications of the questions were narrow and their perceived utility less clear. As noted, the WGQ have been promoted as a tool to disaggregate data by disability. This has been reiterated in guidance for NGO programming. Of course, for this to be possible the questions allow people with disability, or at risk of disability, to be identified. Interview findings show use of the questions was directed towards identification as an end goal, rather than as a step towards disaggregation.NGOs, in the main, are not in the business of compiling large data sets for disaggregation and more complex analysis. An exception may be more sophisticated monitoring and evaluation of large programs. While online survey responses indicated use in monitoring and evaluation, this was not reflected in interviews. From interviews, the primary purpose of using the questions was the identification of people with a disability to increase their participation in program activities. This was, in the main, via rapid data collection using easy to administer tools. Efforts were also localized and limited in scope compared to the studies of prevalence previously noted. Acknowledging the distinction between identification and disaggregation would assist in tailoring guidance to improve use by NGOs.Examples of use that showed clearer program benefits were as a screening tool with subsequent follow up. If the aim is to increase participation in program activities by identifying people who may otherwise be excluded, it is appropriate to use the ‘some difficulty’ rather than ‘a lot of difficulty’ cut off. While the Washington Group recommends the latter for disaggregation of data from censuses and national surveys, the Group recognizes disability is experienced on a continuum . As noteIn terms of analysis, sophisticated methods are not necessarily needed for identification purposes and simple counts will suffice. Interview participants questioned whether their analytical skills were sufficient and suspected they should be doing more. However, this depends on the purpose of analysis and the desired programming goals. Participants faced no difficulties with the analysis required to identify the number of people with disability in their programming area. In summary, consideration of, and clarity on, end use should be the driver of guidance and resource allocation to support use of the questions in programming.As noted, the WGQ are not diagnostic, and identification of people with disability does not equate to identifying needs. The questions also do not in themselves provide information on the barriers individuals with disability face or on the access requirements an individual may have. Clarity in guidance on how the questions may or may not contribute to disability inclusion in programs would be helpful. From interviews, it was evident that expectations of what the questions might contribute to programming were often higher than what may be realistically achieved. There was also confusion over the purpose of the individual questions in the Short Set in particular. Individual questions allow disaggregation by a limited number of activity limitations, such as difficulty seeing or walking, and the response options provide some indication of the level of difficulty experienced. However, this is not their primary purpose.The purpose of the individual questions is to contribute to answering the overarching yes-no question of who has a disability. They are a means to an end that allow disaggregation of data sets and inequality of opportunities to be identified. Similarly, use of the questions in programming needs to be considered as one part of a disability data collection process. The Washington Group questions cannot answer all disability data questions. For example, ensuring reasonable accommodation will still require consultation with the individual concerned. Asking the Short Set to interpret accessibility needs prior to, for example, a workshop is cumbersome in comparison to asking a direct question on accessibility needs. The benefits of asking OPD members the Washington Group questions are not clear when a direct question would suffice. A more discerning approach to what the questions can and cannot contribute to programming, and when, as part of a broader disability data collection process may be more effective.Early iterations of the Short Set and UN census guidance note four core or essential questions and two additional or supplementary questions; that is, questions five on self-care and six on communicating are preferred ,22. The In NGO programming, there is room to consider the staged adoption of the questions. The effective use of the first four questions of the Short Set may identify more people at risk of disability than six questions used poorly. As teams and data collectors become more confident using the questions, the remaining two could be added. The time allocated to training data collectors was also reported as limited in the main. This ranged from half a day in a humanitarian context to an exception of five days, with three days being the norm. Persisting issues, including directly mentioning disability and on-the-fly explanations, suggests that this is insufficient. Particularly when we consider the time required to understand the rationale behind the questions and why disability should not be mentioned; strategies for dealing with known issues and explanations if they are to be provided; and ensuring sufficient time for repeat practice and reflection. Staged adoption of the questions alongside increased attention to avoiding known data collection issues, such as reducing the need for on-the-fly translations, may be more beneficial over time.Despite the challenges organizations are facing using the questions, there are notable positives. Use of the questions to better identify people with disability for inclusion in programs is undoubtedly important. Currently, the WGQ are arguably the most effective and resource efficient tool we have for this purpose. While we did not identify concrete examples of positive impact for people with disability resulting from use in programming, the process of using the questions was contributing to positive change. It was how the WGQ were influencing processes rather than direct outcomes that stood out. That use of the WGQ was changing attitudes was also noted by a Leonard Cheshire and Humanity and Inclusion study conducted at a similar time to this research . While wThe process of adoption and use of the questions was reported as generating new discussions and raising awareness. Use of the questions was elevating the profile of disability inclusion as an issue of focus within offices and projects. Through incorporating the WGQ in their work, teams were learning more about disability and disability inclusion. Similar benefits were also noted in, and through engagement with, communities where organizations worked. Using the questions in communities was generating conversations about disability that program teams had not had before. The process of using the questions, however imperfectly that may be, was reported as contributing to changing attitudes and addressing stigma. In terms of disability inclusive programming, and while acknowledging the challenges of attributing impact, any such outcomes can only be desirable.Our research drew on a small sample of respondents and we are cautious of making broad assumptions. We deliberately focused on organizations that had used the questions and the majority of these had a prior interest in disability inclusion. It is noteworthy that early and motivated adopters faced difficulties using the questions and in assessing what the questions could contribute. If these organizations are facing challenges, it is reasonable to expect organizations new to disability inclusion to face further barriers to adopting and sustaining use of the questions in their work.In terms of disability inclusion, uptake of the WGQ in non-government programming is encouraging. Efforts to identify and increase the participation of people at risk of disability in development and humanitarian programming are clearly welcome and much needed. While we were not able to identify direct outcomes for people with disability arising from use of the questions, the process of using the questions was contributing to change. Examples of positive change were not only within offices and program teams but, for some, extended to host communities.In NGO guidance, it is important to acknowledge that end uses will likely differ from the Washington Group’s original purpose. As noted, flexibility is required. If the aim is advocacy to government, it may be preferable to use the questions in line with the Group’s recommended protocols to ensure consistency. For other programming purposes and aims it may not. Differences should be recognized in guidance and considered in program design. For example, inclusion of ‘some difficulty’ in analysis may increase utility. A staged introduction of use of the Short Set may allow more time for improving rigor in data collection, particularly with teams unfamiliar with disability. While using the full six questions may be preferred, ineffective use of any of the questions is unhelpful.A flexible but considered approach to how the WGQ are used in NGO programming may help better demonstrate possible contributions to impact, an area that warrants further inquiry. In turn, this may promote adoption beyond individual champions to across institutions and by organizations that are yet to actively consider disability inclusion. Currently, it is this process of adopting the questions that shows the most promise for improving disability inclusion in NGO programming.


Purpose: Delineation of subtle lesions in magnetic resonance imaging (MRI)-negative patients is of great importance in preoperative epilepsy evaluation. The aim of our study was to explore the diagnostic value of the novel fluid and white matter suppression (FLAWS) sequence in comparison with a voxel-based MRI postprocessing morphometric analysis program (MAP) in a consecutive cohort of non-lesional patients.Methods: Surgical candidates with a negative finding on an official neuroradiology report were enrolled. High-resolution FLAWS image and MAP maps generated based on high-resolution three-dimensional (3D) T1 image were visually inspected for each patient. The findings of FLAWS or MAP-positive (FLAWS/MAP+) regions were compared with the surgical resection cavity in correlation with surgical outcome and pathology.Results: Forty-five patients were enrolled; the pathological examination revealed focal cortical dysplasia (FCD) in 32 patients and other findings in 13 patients. The positive rate, sensitivity, and specificity were 48.9%, 0.43, and 0.87, respectively, for FLAWS and 64.4%, 0.57, and 0.8, respectively, for MAP. Concordance between surgical resection and FLAWS+ or MAP+ regions was significantly associated with a seizure-free outcome . A positive finding in FLAWS and MAP together with abnormalities in the same gyrus  was detected in 31.1% of patients. FLAWS+ only and MAP+ only were found in 7 (15.5%) and 14 (31.1%) patients, respectively.Conclusions: FLAWS showed a promising value for identifying subtle epileptogenic lesions and can be used as a complement to current MAP in patients with MRI-negative epilepsy. Thirty percent of patients with epilepsy are pharmacoresistant . SurgicaThe fluid and white matter suppression (FLAWS) sequence, a recently developed MRI sequence, was first proposed by Tanner et al.  and deriIn addition to novel imaging acquisition protocols, many postprocessing methods using mature MRI images have been well-developed and utilized in clinical work. These strategies include curvilinear reformatting , 10, texThe aim of this study was 2-fold. Since the detection of subtle abnormalities in conventional MRI-negative epilepsy is both challenging and worth exploring for presurgical evaluation, based on our previous findings, we decided to focus only on MRI-negative patients. In order to improve the study representativeness, the design aimed to consecutively recruit surgical candidates from our epilepsy center. Thus, the first aim was to evaluate the potential value of FLAWS in detecting epileptic lesions in patients with MRI-negative epilepsy. FLAWS is relatively new and has not yet been routinely used in clinical practice. Conversely, MAP is a valuable, well-developed, and practical tool and widely used in >30 centers . Hence, Patients with non-lesional pharmacoresistant epilepsy referred for presurgical evaluation at the Comprehensive Epilepsy Center of Beijing, Xuanwu Hospital, from January 2017 to August 2018, were consecutively included for FLAWS image scanning. The inclusion criteria were as follows: (1) a negative finding on the official neuroradiology report before the multidisciplinary presurgical evaluation conference (MPEC), (2) MPEC recommended resective surgery, (3) a T1-weighted magnetization-prepared rapid acquisition with gradient-echo (MPRAGE) image was available for MAP analysis, and (4) consent for FLAWS scan and participation in this study. The exclusion criteria were (1) age <5 years old at screening, (2) FLAWS was not conducted because of organizational problems, (3) resective epilepsy surgery was not performed, (4) poor imaging quality of conventional MRI or FLAWS image, and (5) the patient was lost to follow-up or had <12 months of follow-up.This study was approved by the Ethics Committee of Xuanwu Hospital and conducted in accordance with the Declaration of Helsinki. Informed written consent was obtained from each patient or legal guardian if the patient was underage.The therapeutic schedule regarding intracranial electrode implantation and the surgical strategy for all patients were discussed at the routine MPEC with neuroradiologists, electrophysiologists, neurologists, neurosurgeons, and researchers dedicated to epilepsy, at the Comprehensive Epilepsy Center of Beijing, Xuanwu Hospital, based on information from semiology, video electroencephalogram (EEG), routine MRI, positron emission tomography, and magnetoencephalography (MEG). Generally, consistent with the guidelines , investiThe clinical routine MRI protocol for epilepsy at our hospital was acquired using a 3.0-T MRI scanner system , including T1WI , T2WI , 2D-FLAIR , 3D-FLAIR , and DWI . An MPRAGE image was acquired on patients who underwent MEG. The MRI image of each patient was re-reviewed at the MPEC. Patients initially reported as “negative” were divided into the “subtly lesional” or the “strictly non-lesional” subgroups depending on whether a subtle abnormality was found at MPEC.An individualized, tailored resection was performed based on the conclusion from converging information by the MPEC. Tissues obtained were sent for pathological examination with experienced neuropathologists dedicated to epilepsy, following the International League Against Epilepsy classification , 22.Patients were followed up by outpatient consultation and telephone interviews at 3, 6, and 12 months postoperatively and at longer intervals thereafter. The surgical outcome was evaluated, according to Engel's classification .FLAWS images were acquired using a prototype sequence with a 12-channel head-neck coil in the 3.0-T MRI scanner system . Two TIs were used to generate the white matter-nulled images  and cerebrospinal fluid-nulled images . The FLAWS image was generated by taking the minimum voxel values of FLAWS1 and FLAWS2. Other details of the scan parameters were previously reported  using the SPM12 toolbox  in MATLAB 2017a  following established methods , 16. ThrSince information from one study could influence the judgment of another, FLAWS and MAP maps were analyzed by different investigators in the following order :Step 1: Independent original FLAWS visual inspection without electroclinical information, MPRAGE, MAP maps, and postoperative computed tomography (CT) image, aiming to report FLAWS-positive (FLAWS+) regions and feature scores. Two experienced radiologists , both with >10 years of experience, reviewed the images. Independent MAP analysis, without electroclinical information, FLAWS, and postoperative CT image, aimed to report MAP-positive (MAP+) regions in junction, extension, and thickness maps. Two experienced epileptologists  with >10 years of experience in epilepsy reviewed the maps.http://www.slicer.org).Step 2: Determination of the relationship between the resection cavity and FLAWS or MAP abnormalities was performed with the registration and segmentation module in 3D Slicer (website: Step 3: A joint re-review of co-registered FLAWS and MAP was conducted to determine concordance or discordance between the FLAWS+ and MAP+ regions (XC and DY).The original FLAWS images were evaluated with regard to the detection of structural abnormalities. Five typical features have been reported as helpful for the visual identification of subtle lesions on MRI and were used in our study: (A), abnormal cortical morphology, including thinning and thickening  and abnormal deep sulci; (B), abnormal cortical signal intensity; (C), blurred GM–WM junction; (D), abnormal signal intensity of subcortical WM; and (E), transmantle sign , 26. Falz-score thresholds of 4, 6, and 4 were used for identifying candidate MAP+ regions on the junction, extension, and thickness map, respectively  visually reviewed the three maps to determine whether a MAP+ region existed, the location of the MAP+ region, and the MAP+ region score on the three maps . The abnormality was reaffirmed by checking with the original MPRAGE image to confirm the MAP+ region.Based on the literature, the ectively , 27, 28.The determination of the relationship between the resection cavity and FLAWS or MAP abnormalities was performed using registration, segmentation, and ruler measurement function in 3D Slicer (KS). First, FLAWS and postoperative CT were registered onto the preoperative MPRAGE using the 3D Slicer individually, which allowed us to analyze the images in the same coordinate system. Registration was unnecessary for MAP maps because they were generated based on MPRAGE. Second, delineation of the resection cavity on the postoperative CT with the segmentation module in 3D Slicer allowed visualization of the surgical margin on MPRAGE, co-registered FLAWS, and co-registered MAP maps, on axial, sagittal, and coronal views. Third, the co-registered FLAWS or MAP was further overlaid with the co-registered postoperative CT to assess the relationship between the resection cavity and FLAWS or MAP abnormalities. The correlation was classified as “concordance” or “nonconcordance,” depending on the distance between the outermost border of the lesion and the surgical resection margin measured using the ruler in the 3D Slicer. Considering the tissue movement around the resection cavity after surgery, “concordance” was only determined if the border was completely within or <8 mm outside of the resection margin, similar to a previous study . If multCo-registered FLAWS and MAP maps were overlaid on the 3D Slicer software to determine whether the FLAWS+ and MAP+ regions were concordant (XC and DY). Concordance was defined at the gyral or lobar level, depending on whether the FLAWS+ and MAP+ regions coexisted in the same anatomic gyrus or the same lobe. Discordance was defined if the FLAWS+ and MAP+ regions are located in different lobes or hemispheres.t and Wilcoxon rank-sum tests were used to compare differences in age and seizure duration between the seizure-free group and the not seizure-free group.Patients were divided into two groups: seizure-free group (Engel I) and not seizure-free group (Engel II–IV), based on the surgical outcome at 12 months. The Pearson chi-square test was used to estimate the correlation of FLAWS or MAP-positive regions with resection to surgical outcome and the difference in age group, gender, resection type, pathology, and invasive exploration before surgery, as well as “MRI subgroups” between the two groups; Fisher's exact test was used when the chi-square test included expected values <5. The The positive rate, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for FLAWS and MAP in the entire cohort. The positive rate was defined as the number of FLAWS/MAP+ cases/the number of the whole dataset. Pearson chi-square test was used to estimate the difference in positive rate between FLAWS and MAP in the overall cohort as well as to estimate the difference between temporal and extratemporal, “subtly lesional” and “strictly nonlesional” subgroups; Fisher's exact test was used when the chi-square test included an expected value <5. True positive was defined as concordance between the FLAWS/MAP findings and resection with good surgical outcome, false positive as concordance between the FLAWS/MAP findings and resection with poor surgical outcome, true negative as non-concordance between the FLAWS/MAP findings and resection with poor surgical outcome, and false negative as non-concordance between FLAWS/MAP findings and resection with good surgical outcome. These definitions were based on those of previous reports in the literature , 30.p < 0.05 (two-sided).All statistical analyses were performed using R, version 3.6.3 , with statistical significance defined as A total of 107 non-lesional patients were screened; of these, three patients <5 years old and 36 patients unsuitable for resective surgery were ruled out, with resective surgical treatment recommended for 68 patients. Out of the 68 surgical candidates, three and eight patients were ruled out either because no MPRAGE was available or written consent was not obtained, respectively. Thereafter, 57 patients signed the consent form and were included in our study. FLAWS was successfully conducted in 52 patients. Four patients who did not undergo resective surgery, two patients who had poor signal on MRI, and one patient lost to follow-up were excluded; thus, a final dataset consisting of the data of 45 patients was used for analysis .Detailed demographics and clinical data are included in p = 0.54). For the 30 patients in the seizure-free subgroup, 27 (90%) were explored with intracranial EEG. All 15 patients in the not seizure-free subgroup underwent intracranial EEG before resective surgery. No significant difference was found regarding the intracranial EEG exploration ratio between the seizure-free and not seizure-free subgroups (p = 0.82).Forty-two patients underwent intracranial EEG, including 36 patients undergoing SEEG implantations and six subdural EEG implantations. Out of the 42 patients explored with intracranial EEG, 27 patients (64.3%) were seizure-free. All three patients who received resective surgery without intracranial EEG were seizure-free. No significant difference in seizure-free outcome was found between patients who received intracranial EEG vs. those who did not , FCD II , FCD III , hippocampal sclerosis , ganglioglioma , gliosis , and non-identifiable abnormalities . The pathology classification was not associated with surgical outcome (p = 0.22).The pathological evaluation revealed FCD I (p = 0.14). p = 0.17).Independent analysis revealed FLAWS+ regions in 22 (48.9%) of the 45 patients. The most frequently detected feature was “C” and the most common feature pattern was “CD.” The positive rate was 33.3% (6/18) and 57.7% (15/26) in the temporal subgroup and extratemporal subgroup, respectively. Although a higher positive rate was shown in the extratemporal subgroup, it was not statistically significant (p = 0.002) , 0.87 , 0.87 , and 0.43 , respectively . In 13 (= 0.002) .n = 6; FCD II, n = 5), ganglioglioma in two patients, gliosis in one patient, and non-identifiable in one patient.Surgical specimens from the 15 patients whose FLAWS+ regions were concordant with resection were pathologically diagnosed with FCD in 11 (73.3%) patients . In the “subtly lesional” and “strictly nonlesional” subgroups, the positive rate was 70 and 62.8%, respectively (p = 1). The positive rate of MAP in patients ≥18 years old and patients <18 years old were 61.3 and 71.4%, respectively, which was not significantly different (p = 0.7).Independent analysis revealed the presence of MAP+ regions in 29 (64.4%) of the 45 cases, of whom two (6.9%) patients had multiple MAP+ regions. In all patients with MAP+ regions, abnormalities were detected in the junction file. Two patients exhibited abnormalities in the corresponding regions in the extension file. No patient showed relevant changes in the thickness file. The positive rate was 61.1% (11/18) and 65.4% (17/26) in the temporal subgroup and extratemporal subgroup, respectively (p = 0.0003) , 0.8 , 0.85 , and 0.48 , respectively. Seventeen of the 20 cases (85.0%), whose MAP+ region was concordant with resection, became seizure-free. One (11.1%) of the nine patients, whose MAP+ region was non-concordant with resection, became seizure-free. Concordance between resection and the MAP+ regions was significantly associated with a seizure-free outcome when compared with the non-concordance group ( 0.0003) .n = 11, FCD II, n = 5), gliosis in two patients, hippocampal sclerosis in one patient, and ganglioglioma in one patient.Surgical specimens from the 20 patients whose abnormalities in MAP were concordant with resection were diagnosed with FCD in 16 (80%) patients . The re-examination of abnormalities in the independent analysis with co-registered FLAWS and MAP maps revealed that 15 (33.3%) patients were positive on both FLAWS and MAP, including gyral concordance in 12 (26.6%) patients . In both patients, the abnormality of GM–WM junction blurring was very subtle and located in a very small region. Therefore, these features escaped the initial visual FLAWS inspection .p = 0.004)  in the overall cohort had a sensitivity, specificity, PPV, and NPV of 0.3 , 1 , 1 , and 0.42 , respectively. Nine of the nine patients (100%) whose FLAWS–MAP gyral+ region was concordant with resection became seizure-free, and one of the five patients (20%) whose FLAWS–MAP gyral+ region was not concordant with resection became seizure-free. Concordance between resection and the FLAWS–MAP gyral+ regions was significantly associated with a seizure-free outcome, compared with the non-concordance group (= 0.004) .n = 4; FCD II, n = 4) and with ganglioglioma in one patient.Surgical specimens from the nine patients whose abnormalities in FLAWS–MAP gyral+ region were concordant with resection were diagnosed with FCD in eight (88.9%) patients  in comparison with a well-developed postprocessing method (MAP) among patients with MRI-negative epilepsy. Our main findings were as follows: (1) FLAWS could detect subtle abnormalities with high specificity (0.87) and low sensitivity (0.43), (2) MAP could detect subtle abnormalities with a higher sensitivity (0.57) and a similar specificity (0.80) as those of FLAWS, and (3) a complementary value from FLAWS existed in MAP-negative patients.The application of FLAWS in epilepsy has been rarely reported. We have previously reported that the sensitivity and specificity of FLAWS in patients with conventional MRI-positive and MRI-negative FCD were 71.9 and 71.1%, respectively . By compIn patients with normal appearance on conventional MRI, lesions are usually subtle and difficult to detect. We found that the most frequent detected single feature was “blurred junction of GM–WM” and the most common feature combination pattern was “blurred junction of GM–WM with abnormal signal intensity of subcortical WM.” Owing to the highly convoluted nature of the cortex, a proportion of certain images may contain a mixture of several tissues, such as WM, CSF, and GM . CSF andThe strategy of suppressing the WM and CSF signals was also employed in the double inversion recovery turbo spin-echo sequence previously developed . Double A direct comparative study between DIR and FLAWS in epilepsy was unavailable. However, one technique study reported that FLAWS was advantageous for having a single inversion pulse preparation and small flip angle gradient-recalled echo readout compared with DIR sequence, which has two inversion pulse preparation and a large flip angle turbo spin-echo readout. Ultra-high field 7 T MRI scans in healthy subjects revealed that FLAWS exhibited more homogeneous WM suppression and better GM visualization than DIR . FurtherIn this study, MAP revealed subtle abnormalities in 64.4% of patients, a finding generally consistent with previous literature , 40, 41.MAP generates junction, extension, and thickness maps after normalization, segmentation, the convolution of individual patient's image, and the comparison of individual patient's image with a database of normal controls. Through these steps, the output maps indicate blurring or aberrant GM beyond the cortical ribbon with hyperintensity , 15 and In our study, MAP alone had a higher sensitivity than FLAWS alone, and the exclusive yield was higher with MAP than in FLAWS . In addition, two additional patients, negative in the independent visual FLAWS analysis, became positive under the MAP guidance. The abnormalities on the FLAWS images of these two patients were located in a very limited region and consisted of a few connected gyri , which lIn addition to the superiority of MAP, there are two other crucial findings from our study. The first was the complementary value of FLAWS for MAP analysis. Seven patients had abnormal findings in FLAWS analysis only, which indicated the unique value of the FLAWS sequence. In the comparison of the FLAWS image with the MPRAGE one, as shown in Another important finding was the revelation of a high gyral FLAWS–MAP concordance. Despite the difference in methodology between these two techniques, 31.1% of patients showed gyral FLAWS–MAP concordance. Considering the surgical outcome and pathological findings, we believe that common structural changes rather than coincidences are underlying these co-detected abnormalities.Abnormalities on MAP maps represent the deviation from a normal distribution; therefore, suprathreshold regions may be caused by registration errors, imaging artifacts, or normal variants. Assessment of subtle but visually identifiable changes using T1, T2, and FLAIR images is the usual method for validating MAP results , 41, 44.In general, patients with negative MRI results have poorer postoperative outcomes and may not be referred for surgical treatment. Accurate detection and delineation of lesions cannot be overstated. Rapidly evolving postprocessing methods using currently available mature MRI sequences such as MPRAGE, T2, and FLAIR have augmented and improved the detection of subtle lesions, including FCD and other pathological diseases. However, the findings of complementary yields by visual FLAWS inspection to MPRAGE-based MAP analysis and the high PPV of FLAWS–MAP gyral+ abnormalities observed in our study suggest novel MRI sequences still worth exploring.Our study has several limitations. Our study is limited in that only postoperative CT was available. Further studies with postoperative MRI are needed to verify our findings. A voxel-based morphometric statistical analysis using FLAWS images to compute the deviation of individual patient's image to normal distribution was not performed, because a large number of age-matched healthy volunteers would be needed. A comparison of FLAWS to T2 or FLAIR-based MAP analysis was also not performed in our study, because only high-resolution MPRAGE images were available in our hospital. Further studies are warranted to prompt more comprehensive results. Furthermore, normal pediatric databases were not available in this study. Although no significant difference was found between the age ≥18 and age <18 groups in the present study, age-specific normal databases for voxel-based morphometric analysis are advisable.FLAWS, as a new tool, showed a promising value for identifying subtle epileptogenic lesions. It can also be used as a complement to current MAP in patients with MRI-negative epilepsy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Xuanwu Hospita. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.KS analyzed the clinical data, reviewed the FLAWS and MAP with postsurgical CT image, and wrote the manuscript. TY, LQ, and DN performed the surgical treatment. DY reviewed the MAP maps and revised the manuscript. ZR collected the FLAWS data. XW reviewed the MAP maps. YZ revised the manuscript and reviewed the FLAWS image. XC collected the FLAWS data, reviewed the FLAWS image, and revised the manuscript. JX revised the manuscript. NC reviewed the FLAWS image. RG and YL reviewed the literature. KY participated in the data statistical analysis. TK designed the FLAWS sequence. GZ conceived the study, supervised the data analysis, and revised the manuscript. All authors contributed to the article and approved the submitted version.TK is a full employee of Siemens Healthcare AG Switzerland. The remaining 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.


Super-resolution optical imaging is a consistent research hotspot for promoting studies in nanotechnology and biotechnology due to its capability of overcoming the diffraction limit, which is an intrinsic obstacle in pursuing higher resolution for conventional microscopy techniques. In the past few decades, a great number of techniques in this research domain have been theoretically proposed and experimentally demonstrated. Graphene, a special two-dimensional material, has become the most meritorious candidate and attracted incredible attention in high-resolution imaging domain due to its distinctive properties. In this article, the working principle of graphene-assisted imaging devices is summarized, and recent advances of super-resolution optical imaging based on graphene are reviewed for both near-field and far-field applications. Since shickness , opticalhickness , electrihickness , and thehickness ,7 properhickness , growth hickness , deposithickness ,11,12, ahickness ,14,15.One of the most important properties of graphene is that the complex conductivity can be dynamically tuned by external parameters ,17,18 suλ⁄2NA . This intrinsic limit, also known as diffraction limit [After the first microscope was invented and named as well as applied to observe cells successfully in the 17th century, microscopic techniques remain the most widespread imaging method and always play an irreplaceable role in the research field of nanotechnology and biotechnology, especially in bioscience , owing ton limit ,30, has on limit ,32,33; fon limit ,35,36, hon limit ,38,39, aon limit ,41,42,43on limit ,45,46,47on limit ,50,51,52on limit ,54,55; pon limit ,57,58, aon limit ,60. It iAchieving super-resolution imaging means to overcome the diffraction limit originated from the exponential decay of the evanescent waves which carry the high spatial frequency information of the objects. In essence, overcoming the fundamental limit means magnifying the evanescent waves directly or converting evanescent waves to propagating ones and further providing magnification. Graphene can significantly enhance evanescent fields due to the fact that its conductivity can be tuned in the infrared and terahertz (THz) regions ,62.T, the radian frequency w, the momentum relaxation time τ, and the chemical potential Among all these amazing properties of graphene, the one that is most worthy of mention is that the surface conductivity of graphene can be tuned via external parameters ,63. The d doping ,68. The ications ,70,71.Moreover, since surface plasmons (SPs) have been experimentally demonstrated in graphene , graphenλ⁄11-resolution via graphene-enhanced NSOM in 2014 [For near-field applications, graphene has been integrated with NSOM, superlens, and wire medium, and therefore can provide magnification of the evanescent waves for achieving super-resolution imaging ,87,88 vi in 2014 . A sharpIn 2015, Forouzmand and co-workers proposed two kinds of novel graphene-loaded wire medium (WM) slab structures that were suitable for dual-band or tunable broadband super-resolution imaging in near field ,82. FiguAnother graphene-assisted subwavelength imaging device was reported in 2017  which reA fast-paced graphene-based near-field optical microscopy (GNOM) for overcoming the diffraction limit was proposed by Inampudi and coworkers who utilized the electronic scanning property of graphene gratings  . Figure In 2018, Liu and co-workers introduced a graphene sheet as an ultrathin nonlinear negative reflection lens for achieving super-resolution imaging based on four wave mixing (FWM) process in the terahertz regime  thanks tIn far field, graphene has normally been integrated with a hyperlens. Surface plasmons can help convert the evanescent waves to the propagating ones and therefore provide magnification for achieving super-resolution imaging via the tunable conductivity and the coupling of graphene plasmons. In 2013, Zhang and coworkers designed two kinds of different hyperlenses  composedd⁄cosθ and d(r + t))⁄r for the triangle-shaped and cylindrical hyperlenses, respectively. In addition, it is worth noting that the layered structures can achieve propagation of the evanescent waves for a fixed wavelength by manipulating cμ, as shown in The working mechanism is identical for these two kinds of hyperlenses, which relies on the fact that the hyperbolic dispersion curve can amplify and support the propagation of the evanescent waves straightly along the normal direction of the layered structures and form two imaging spots at the output plane under the condition of In 2016, Yang and coworkers proposed a graphene nanocavity on metasurface structure (GNMS)  to excitTo further study the property of the GNMS device, the function of the grating and the effect of the structural parameters on the graphene plasmonic interference pattern were discussed. According to the results, one can learn that the period of the graphene plasmonic interference pattern is 52 nm when the wavelength of the incident light is 7 μm. When the GNMS device is integrated with PSIM, an imaging resolution of 26 nm can be achieved because of the graphene plasmons with deep sub-wavelength. 2/Ag/SiO2 multilayer design. From Moreover, the same group proposed another elegant design to realize wide-field optical imaging based on a hybrid graphene on metasurface structure (GMS) model . Figure In this work, it is crucial to utilize the most significant feature of graphene plasmons, which is the ultra-high wave vector, to combine the model of GMS with the PSIM method and further achieve super-resolution imaging. The authors employed the finite-difference time-domain (FDTD) method to model and simulate the GMS structure and found that the standing wave of surface plasmons (SW-SPs) with an 11 nm period can be achieved on graphene. Furthermore, when the GMS structure was applied in the PSIM method, they found that an imaging resolution of 6 nm could be obtained for a 980 nm illumination wavelength which was improved 39.6-fold in comparison with the conventional microscopy technique with resolution of 283 nm. Additionally, the resolving capability of GMS–PSIM system was acquired by imaging two point objects separated by 6 nm. The simulation results are shown in In addition to the tunable surface conductivity and graphene plasmons, graphene has been introduced to the applications of electron microscopy to achieve super-resolution imaging of wet cells by utilizing the impermeable and conductive properties. More important bioimaging applications can be enabled using graphene-based nanomaterials  and their derivatives ,93,94. WGraphene oxide (GO) has also found extensive applications in integrated photonics ,97 and tTo conclude the review, the working principles, implementations, and performances of the super-resolution imaging integrated with graphene/GO have been comprehensively summarized. Due to the most useful property that the surface conductivity of graphene can be rapidly tuned by external parameters, graphene is capable of enhancing the evanescent waves. The present review strongly suggests that graphene has great advantages in super-resolution imaging in the infrared and THz regions both in near field and far field. However, the use of the graphene for super-resolution imaging is still at a very early stage, which means that we still need a lot of time and efforts to explore its full potential experimentally. In our opinion, thinner, faster, and more reliable imaging devices using graphene-assisted components are highly desired for various practical applications and therefore, more efforts should be made to develop related devices. More flexible components are also needed to meet the increasing future demands. Moreover, thorough investigations on structural parameters are necessary for studying their impact and working mechanisms. Given the progress in both the theory and fabrication of the graphene-assisted materials, there is no doubt that more methods and devices on super-resolution imaging with graphene will be developed to meet the increasing demanding of more practical application requirements in the near future.