Answering questions before a learning episode—“prequestions”—can enhance memory for that information. A number of studies have explored this effect in the laboratory; however, few studies have examined prequestions in a classroom setting. In the current study, the effects of prequestions were examined in an undergraduate course in chemical engineering. At the start of several class meetings, students were provided with a prequestion to answer about the upcoming lesson, and then were asked to provide ratings of confidence in their answers, familiarity with the content in the prequestion, and how much of the assigned reading they had completed. At the end of class, students were given the same question again (postquestion), along with a different question from the same lesson (new question). On a quiz at the end of each week, students were given the postquestions and new questions again, in addition to never-before-seen questions (quiz-only questions) from the same lessons. Performance on questions at the end of class revealed no difference in performance for postquestions vs. new questions. Although weekly quiz performance revealed an effect of retrieval practice—superior memory for material tested at the end of class (postquestions and new questions) compared to material not tested (quiz-only questions)—there was no difference in weekly quiz performance on postquestions vs. new questions. These results suggest that retrieval practice is beneficial to learning in the classroom. However, prequestions do not appear to enhance learning, nor to enhance the effects of retrieval practice.The online version of this article (doi:10.1186/s41235-017-0078-z) contains supplementary material, which is available to authorized users. An important question in education is how to improve students’ learning of course concepts. Asking students questions over what they are learning—i.e., retrieval practice—is an effective way to enhance retention of concepts. However, cognitive psychology research has not always provided clear and concrete recommendations for how instructors can incorporate this technique into their teaching. The current study shows that “clicker” questions administered at the beginning and end of class can provide a straightforward and effective way to enhance students’ learning in chemical engineering. In particular, material that was tested at the end of class was remembered 30% better on a later review quiz, relative to material from the same lesson that was not tested. Asking questions at the beginning of class—i.e., “prequestions”—and repeating them at the end of class, however, did not further this benefit. Thus, when instructors are faced with the question of when to incorporate clicker questions into a given day’s class—at the beginning, at the end, or both—the current results suggest that such questions would be best placed at the end of class. These results provide a simple yet effective way that instructors can incorporate retrieval practice into their teaching.testing effect, or retrieval practice , and the other half read each segment without receiving pre-questions . On the final test, the prequestion group typically outperforms the control group , and a modest but significant advantage in experiment 2b . Thus, contrary to the results of laboratory studies where the benefits of prequestioned information over non-prequestioned information have been quite large—e.g., 25% in the study by Richland et al. (d = 1.70)—these effects appear to be much smaller in classroom settings.Indeed, in the 10 years since the publication of the practice guide, the potential of prequestions to enhance learning in educational settings has remained largely unexplored. To date, only one known study has explored prequestions in a classroom setting. McDaniel, Agarwal, Huelser, McDermott, and Roediger   believed to be responsible for these effects are less likely to be engaged. Theoretical discussions of the prequestion effect have included the  possibilities that prequestions stimulate curiosity  class period, students may have difficulty sustaining attention and noticing information that is relevant to a prequestion that was asked at the beginning of class. Even if curiosity to know the answer is high, this rather lengthy duration of time may introduce interruptions in the processing of the prequestion and the ease with which students can remember it and connect it to the relevant information in the lesson.In the current study, we set out to provide additional data on the effectiveness of prequestions in classroom settings. In a college-level course on chemical engineering, students were asked a question at the beginning of several class meetings that pertained to that day’s lesson. At the end of class, they were asked the same question again, in addition to a different question from the same lesson. Consistent with laboratory studies on prequestions, we did not provide students with feedback of the correct answers at the time of the prequestions. The answer to a given prequestion was always contained in the class lesson that immediately followed, and to learn the answer students needed to discover it during class. This was somewhat different from the study by McDaniel et al. , in whicGiven the variability among students in classroom environments, we also explored the potential role of individual differences in the prequestion effect. We collected information about students’ academic achievement ), in addition to their confidence in their answers to the prequestions, familiarity with the information in the prequestions, and how much of the assigned reading they had completed prior to class. Such individual differences have not been examined in any of the known research on prequestions, but could be of theoretical and practical importance. In particular, the idea that prequestions provide a metacognitive reality check that reduces students’ overconfidence could be tested by examining the relationship between students’ confidence in their answers to the prequestions and their later accuracy on those questions at the end of class. A negative relationship between confidence and later accuracy (other factors controlled) might offer some support for this notion, in that students who are less certain about their knowledge of the concept in the prequestion are more likely to learn and retain the answer to that question when it is encountered in class.Abundant research on retrieval practice shows that testing students over material they have encountered boosts memory retention . The study protocol was reviewed and approved by ISU’s Institutional Review Board (IRB). At the beginning of the semester, a researcher visited class and informed students about the study. All students were invited to participate by providing informed consent that allowed their scores on the course-related activities to be analyzed and information about their grade point average (GPA) to be collected for research purposes. Throughout the semester, three students dropped the course and 12 students declined to have their data included in the study, leaving 62 students in the sample.The course was a Material and Energy Balances course for second-year chemical engineering majors. The course covered three content modules: (1) material balances, (2) volumetric properties and multiphase systems, and (3) energy balances. Following the content modules, there were two modules devoted to case studies. The course met three times per week for 50 minutes over a 15-week semester. There were ten class meetings dedicated to instructional activities for each of the three content modules. Other class meetings were dedicated to scheduled exams, review of information following exams, and case studies.The course was structured using the team-based learning instruction method  consisted primarily of hands-on problem-solving exercises or scheduled activities related to exams. Thus, the prequestions took place on days that were well-suited for the acquisition of new information that students needed for later stages of the course.For each of the 15 target class meetings, a set of three questions was selected to align with the content for that day. These questions assessed knowledge of concepts that were included in the assigned readings for each class meeting, although the exact questions themselves were not included in the readings. Each set of three questions contained a prequestion, a new question, and a quiz-only question. The prequestion was asked at the beginning of class and repeated at the end of class, whereas the new question was asked only at the end of class. This allowed us to measure retention at the end of class for prequestioned vs. non-prequestioned information. To explore whether prequestions enhance the effects of retrieval practice, an online quiz was given to students at the end of each week. This online quiz contained both the prequestion and the new question from that week, along with a third question from the same lesson that had not been asked before—i.e., the quiz-only question. This allowed us to explore the delayed effects of questions that had been asked at the beginning and end of class (prequestions), versus questions asked only once at the end of class (new questions), and to compare performance on these questions to performance on questions from the same lesson that had not been asked before (quiz-only questions). Better performance on new questions vs. quiz-only questions would demonstrate the benefits of retrieval practice. Better performance on prequestions vs. new questions would demonstrate that the effects of retrieval practice are bolstered by providing an opportunity to answer the questions at the beginning of class.In this real classroom environment, questions could not be randomly assigned or counterbalanced across question type. Therefore, we ensured that the relevant properties of the questions were controlled to the extent possible. All of the questions were selected from the AIChE Concept Warehouse . The time limit for responding was set so that once the response rate reached 90%, the remaining students had 5 s to input an answer. Most prequestion responses were completed in under one minute. No feedback was provided about the correct answer to the prequestion or the distribution of responses. Immediately after the prequestion, students were presented with individual slides assessing their confidence in their answers, familiarity with the concept in the prequestion, and completion of the reading assignment. The confidence question inquired “How confident are you in your answer?” with response options A) very confident, B) confident, C) somewhat confident, or D) not at all confident. The familiarity question inquired “Which statement best describes your familiarity with the question you just answered?” with response options A) very familiar: I knew this concept before class, B) somewhat familiar: I have seen this concept, but I do not remember the details, C) Somewhat unfamiliar: I think I have seen this concept, but I do not remember it, or D) very unfamiliar: this concept is new to me. The reading assignment question inquired “How much of the reading assignment did you complete prior to today’s class?” with response options A) all of it, B) more than half, C) less than half, or D) none of it. Students answered each of these questions one at a time with individual clickers.A schematic of the procedure is shown in Fig. Near the end of the team-based learning session, teams reported their answers to the class and the instructor provided closing remarks to summarize and conclude the team activities. During the last segment of the class period , the instructor presented the prequestion again, with the answer choices arranged in a new, unique order that was different from what students saw at the beginning of class. After students answered the question with their clickers, the confidence question was presented in the same format as at the beginning of class. After all confidence responses were collected, the instructor presented the correct answer to the prequestion and provided a brief explanation. Next, the new question was presented, followed by the confidence question. After all responses were collected, the instructor presented the correct answer to the new question and provided a brief explanation.The slides for the prequestion and new question sequence were kept in a separate file from the slides used for the lesson. Although students were provided access to the lesson slides outside of class, they were not directly provided with the questions that were used as prequestions and new questions. Aside from the class period in which these questions were administered, students were provided with access to these questions again only on the weekly quizzes.Each week except for those that contained exams, students were required to complete a weekly quiz on the online course management system (Blackboard Learn). This quiz contained the prequestion, the new question, and the quiz-only question, in that order, for any of the class periods during that week that implemented the prequestion routine. The answer choices for the prequestion and the new question were arranged in an order that was different from any previous order in which they appeared (when they were presented in class). Additional questions designed to be more challenging were included in the weekly quiz, at the instructor’s discretion, and these other questions  appeared after the prequestion, new question, and quiz-only questions. The quiz was released to students immediately after the last class meeting on Friday of each week, and was due by Sunday at 10 pm.The online quizzes were not graded for correctness. Students received one point per answer for the first attempt for every quiz question that they answered by the deadline. Once a student submitted the quiz, they received a performance report that showed each question, their response to each question, the correct response to the question, and feedback from the instructor to explain the reasoning behind the correct answer. Once a weekly online quiz was released, it remained available to students for the duration of the semester and students could return to the quizzes as often as they wished to review the content. For data analysis purposes, we assessed quiz performance on only the first attempt at answering the prequestions, new questions, and quiz-only questions.The first of the 15 target class meetings (during week 1) was used as a practice opportunity. During this meeting the instructor familiarized students with the in-class question procedure, ensured that clickers were working properly, and reminded students to complete the first weekly quiz before the deadline. Data from this practice session were not analyzed, leaving 14 class meetings (beginning in week 2) that were entered into the analyses.Students’ responses to the prequestions, new questions, and quiz-only questions were scored as either correct or incorrect. Students’ responses to the beginning-of-class questions assessing confidence, familiarity, and reading were re-coded  to numeric ordinal responses  such that higher numbers corresponded to greater degrees of confidence, familiarity, and reading. For each student we calculated the proportion of questions answered correctly on both the in-class questions and the online weekly quiz questions across the 14 target lessons. We also calculated students’ mean confidence, familiarity, and reading ratings across these lessons.01 is used to express the probability of the data given the null hypothesis (model 0) relative to the alternative hypothesis (model 1). Jefferys , we also report the Bayes factors for all nonsignificant comparisons. Unlike NHST that does not allow one to measure support that there is a true null effect, Bayes factors allow one to provide evidence for one model relative to another model. In this case, model 0 is the null hypothesis that there is no difference between conditions, and model 1 is the alternative hypothesis that there is a difference. The notation BFJefferys  providesFor in-class questions, data were included for a given lesson only if students completed all three questions for that lesson—the prequestion at the beginning of class, the same question repeated at the end of class (referred to here as the postquestion), and the new question appearing only at the end of class. Across students, the average number of lessons  that received responses on all three questions was 11.37 (standard deviation (SD) = 2.48). Three students did not provide enough responses to contribute data for the three-question set associated with at least one lesson, resulting in 59 students entered into the following analyses.For each student, we calculated the proportion of questions answered correctly out of the lessons for which that student responded to all three questions. Given differences in the overall mean difficulty (from the AIChE Concept Warehouse) across prequestions  and new questions  associated with these lessons, we entered question difficulty as a covariate in the analyses comparing question types.t(58) = 4.01, p < 0.001, d = 0.74), reflecting some prior knowledge of the material at the time of the prequestions.Figure t(58) = 4.50, p < 0.001, 95% confidence interval (CI) , d = 0.65). However, there was no significant difference in performance at the end of class on the postquestions vs. the new questions (t(58) = 1.52, p = 0.13, 95% CI , d = .35, BF01 = 3.20ts < 1.43, ps > 0.15, BFs01 > 3).Comparison of performance on prequestions vs. postquestions indicated that students improved significantly at answering the same question from the beginning of class to the end of class  receiving responses to all of these questions was 10.51 (SD = 2.65).t(58) = 7.04, p < 0.001, d = 0.66. At the end of class, confidence ratings were also higher for postquestions compared to new questions  = 8.92, p < 0.001, d = 0.88).On average, students found the prequestions to be somewhat familiar, with an average rating of 3.11 (SD = 0.43). The average rating for out-of-class reading was 3.06 (SD = 0.66), indicating that students completed just over half of the assigned reading per lesson. Confidence ratings increased significantly from prequestion  to postquestion : R2 = 0.20, F = 2.64, p = 0.034). The only predictor to emerge as significant was prequestion accuracy  = 0.14, p = 0.001), indicating that students were more likely to answer the postquestion correctly if they had answered the prequestion correctly. No evidence emerged that prequestion confidence, or any of the other predictors, was significantly related to postquestion accuracy. Bayesian analyses, however, revealed weak evidence in favor of these null effects (BFs01 < 3).A multiple regression analysis was conducted with postquestion accuracy as the dependent variable, and five predictors: accuracy on the prequestion, confidence on the prequestion, GPA, familiarity with the prequestion, and out-of-class reading. Table ts < 1.30, ps > 0.20), with Bayesian analyses again suggesting weak evidence in favor of the null effects (BFs01 < 3).The same regression analysis was conducted with new question accuracy as the dependent variable. None of the predictors accounted for a significant amount of variance in new question accuracy , had seen once before (new questions), or had never seen before (quiz-only questions). The benefits of retrieval practice would be reflected in an advantage of new questions over quiz-only questions. If prequestions add to this benefit, then weekly quiz performance would be greater for postquestions compared to new questions.Analysis of performance on the weekly quiz questions required that students complete all three questions on the weekly quiz, in addition to the previous in-class questions corresponding to those same lessons. For example, if a student completed the three online quiz questions for a given lesson , these data were only included if the student had also completed the three in-class questions for that same lesson . Across students, the average number of lessons  receiving responses on all six questions was 10.44 (SD = 2.82). Five students did not provide enough responses to contribute data for the six-question set associated with at least one lesson, resulting in 57 students entered into the following analyses.For each student, we calculated the proportion of questions answered correctly out of the lessons for which that student responded to all six questions. Given differences in the mean difficulty (from the AIChE Concept Warehouse) across postquestions , new questions , and quiz-only questions  associated with these lessons, we entered question difficulty as a covariate in all of the following comparisons.t(56) = 6.38, p < 0.001, 95% CI , d = 1.35) and better on postquestions compared to quiz-only questions (t(56) = 4.03, p < 0.001, 95% CI , d = 0.93). However, students did not perform better on postquestions compared to new questions (t(56) = 0.77, p = 0.44, 95% CI , d = 0.16, BF01 = 7.20), indicating that prequestions did not enhance the benefits of retrieval practice. Question difficulty did not significantly influence performance in any of these comparisons .Figure The current study contributes new data on the effects of prequestions in classroom settings. Asking a question at the beginning of class did not enhance learning of that information, relative to other information from class that was not prequestioned. These results are consistent with those of McDaniel et al.  showing Nor do they appear to enhance the effects of retrieval practice. On the weekly quizzes, the benefits of retrieval practice were apparent, with performance highest for material that was tested at the end of class compared to material that was not tested. Performance did not differ, however, for material that was tested at the beginning and end of class compared to material that was tested only at the end.Thus, information that was tested twice was not remembered better on a later quiz, compared to information that was tested only once. This could be due in part to the fact that feedback was not provided at the time of the prequestion. When students are uncertain about the correct answer, the most potent learning experience is likely to come from corrective feedback. Assuming that the learning gains from practice questions would be enhanced when feedback is provided


We report command electro-optical switching on photolithographically-patterned graphene into a high-density electrode pattern for a high-transmission in-plane-switching (IPS) liquid crystal device. A highly-effective liquid crystal photoalignment method is used to maximize the field-driven optical contrast of a prototyped device. A non-contact and low-temperature photoalignment allows delicate surface treatment required for successful processing of graphene layer into an IPS electrode structure. Electro-optic performance of the graphene-based single pixel laboratory IPS prototype demonstrates the application potential of graphene for liquid crystal electro-optic devices with complex and high-definition electrode patterns. Large Young’s modulus and elasticity of atomically thin graphene5 and graphene oxide6 sheets enable extraordinary flexibility7 and consideration of these materials for flexible applications with large deformations8.Development of transparent electronic and electro-optical (EO) devices is a topic of major interest in modern science and technology. Good electrical conductivity, excellent optical transmittance and mechanical flexibility generate continuous interest in graphene as a candidate transparent conductive material for applications in a large variety of optoelectronic and photonic devices including solar cells, light emitting devices, photodetectors, smart windows, and liquid crystal devices (LCD)14. Consideration of graphene and graphene oxide transparent electrodes has been extended for applications in voltage controlled liquid crystal (LC) terahertz phase shifters18, polymer dispersed liquid crystal (PDLC) devices on rigid glass and flexible plastic substrates20, and flexible plastic contact lenses21. These works have demonstrated suitability of transparent graphene electrodes for application in LC EO devices, and at the same time have indicated technical challenges related to mechanical processing and handling of graphene. Nearly all LC EO devices require accurate control of the average orientation of the LC molecules, known as LC director, which is typically defined by the alignment layer deposited above electrically conducting electrodes. Traditionally, the alignment layer is a thin polymeric film which is mechanically rubbed in order to define the direction of desired LC orientation23. Being an atomically thin carbon film, graphene by its nature is much more vulnerable to irreversible mechanical damage which may be caused during the rubbing process compared to much thicker and mechanically more robust films of solid inorganic transparent conductive oxides. Additionally, graphene can be delaminated from the substrate as result of a contact with n-methyl-2-pyrrolidone (NMP) organic solvent which is a major component of all solutions of commercial alignment layer materials such as polyimides (PI)17. A partial solution of the alignment issue for LC on graphene electrodes could be to use a water soluble poly (PVA) as alignment layer12. However, challenges to align the LC using rubbing of either PVA or PI alignment layers require to consider non-contact alignment for the best optical contrast ratio (CR) and EO performance. Such techniques are photoalignment in the case of planar alignment and polymer stabilization for vertical alignment (VA)26.Graphene-based electronic devices have stimulated a continuous interest for application of graphene as a transparent conductive electrode in LC EO devices28. The photoalignment is a well-known technique for LC alignment via an anisotropic surface interaction capable of a large area uniform alignment for display applications31. At the same time, photoalignment has a unique potential for creation of complex LC orientation patterns implementing lattices of topological features for active control of soft matter LC systems32. Until now an azo dye LC photoalignment has only been demonstrated on a non-patterned porous graphene layer for terahertz LC phase shifter16.The photoalignment layer is a thin film of a photosensitive material capable of creating a high-quality orientational ordering of LC director on its surface. The orientation direction of LC director is defined by irradiation treatment with linearly polarized light having a narrow wavelength spectrum and accurately controlled polarization axis with respect to the surface of the substrate34, “fishbone” VA26, or fringe field switching (FFS)36 LCD modes. We produce a high-quality graphene-based single pixel laboratory LCD prototype using commercially available photoalignment material. Direct comparison of prototypes with identical design demonstrates better optical transmittance and equivalent EO performance of IPS device with graphene electrodes compared to reference device with ITO electrodes.In this work we report in details on development of the fabrication process capable of producing a high-resolution patterns of interdigitated electrodes for modern in-plane switching (IPS)38. A single layer graphene film absorbs from 0.6% (at 780 nm) to 1.8% (at 380 nm) of incident light, as shown on Fig. 13.Graphene films were grown on Cu films using a chemical vapor deposition (CVD) process and transferred onto a quartz glass substrates using a thin PMMA film as the supporting substrateThe graphene-based LC device was fabricated from glass substrates deposited with a single layer graphene film in the center, as shown on Fig. In order to be able to apply electric field to the graphene layer the substrate was additionally deposited with ITO conductive layer covering the right and left edges of the graphene film. The center part of the graphene film and the substrate was protected by a shadow mask splitting the ITO layer into right and left parts which are electrically connected only through the graphene layer in the center of the substrate. Afterwards, both ITO and graphene films on the substrate were patterned using a photolithographic process with a positive photoresist and photomask to produce a single graphene IPS pixel with ITO contact bus lines, as illustrated on Fig. Durability of ITO allows to define the alignment direction of LC easily by mechanically rubbing the surface of alignment layer. However, a high sensitivity of graphene to direct mechanical impacts such as rubbing makes it nearly impossible to apply reliably industry’s standard PI alignment layer materials for a high-quality planar alignment of LC in a cell with graphene electrodes. Even a minor mechanical buffing can create substantial irreversible damage of the integrity of graphene layer. Therefore, we create a high-quality planar alignment of the LC via a non-contact photoalignment method using a commercial polymerizable photoalignment material LIA-01 (from DIC Corporation). The solution of LIA-01 contains potentially harmful to graphene NMP solvent. However, a non-contact alignment process and low temperature processing allow gentle coating of inner surfaces of both substrates successfully yielding a properly functioning IPS electrode structure. The orientation of the LC is chosen to be at 15° with respect to IPS electrodes and was optically generated in assembled empty cells producing perfectly matched alignment on both top and bottom substrates simultaneously, which is a distinct advantage of the photoalignment technique.Characterization of manufactured IPS prototypes consisted of quantitative electro-optical measurements on optical bench, and qualitative observations between crossed polarizers and under the polarizing optical microscope (POM). The performance of graphene-based IPS prototype was evaluated by comparing its static transmittance voltage response (TV curve), response time, and contrast ratio (CR) with a reference ITO-based IPS prototype of identical design. A commercial nematic liquid crystal (NLC) mixture TMS83700 (from HCCH) having positive dielectric anisotropy (Δε = 10.6), low birefringence (Δn = 0.1), and low viscosity (γ = 0.063 Pa·s) is used in all characterization experiments with both types of manufactured IPS devices. A He:Ne laser (633 nm) is used as a light source for electro-optic measurements, and a white light for observations between crossed polarizers.39. However, since the thickness of both ITO and graphene electrodes is much smaller than the cell gap thickness and dimensions of the IPS pattern this difference in driving voltage and maximum brightness is indistinguishable in current experiment. Numerical analysis confirms that visible separation between TV curves from ITO and graphene IPS prototypes has the strongest contribution from electro-optic effect of minor structural imperfections in produced IPS electrode patterns, and a weak contribution from a small deviation in the alignment orientation of LC with respect to electrodes in different cells.The TV curve of graphene IPS prototype is consistent thorough multiple switching cycles and matches well with the TV curve of the reference ITO IPS prototype. Normalized TV curves of graphene and reference ITO IPS cells measured with 1 kHz square waveform AC voltage driving match closely, as shown on Fig. The response time of both graphene, and reference ITO prototypes is measured for switching between 1 V and 5.66 V corresponding to the state of maximum transmittance, and calculated as a change of transmittance between 10% and 90%. Experimentally measured average turn-on switching time of the graphene IPS cell is 20.1 ms, which is 10.7% smaller compared to the reference ITO cell (22 5 ms). The average turn-off switching time of graphene IPS cell is 23.8 ms, and is 4.4% larger compared to the ITO IPS cell (22.8 ms). The liquid crystal surface anchoring strength on graphene substrates is comparable to that of ITO substrates manifested by the negligible difference in response times of the two cells as shown in Fig. γ, cell gap thickness d, and twist elastic constant K22, and analytically can be approximated as follows34:K22 = 6.8 pN for NLC mixture TMS83700 demonstrates that observed difference in turn-off response time corresponds to a difference in the cell gap thickness of 2.2%. Empty prototype cells had a small deviation of the cell gap thickness of up to +/− 0.2 µm or +/− 5.7%. Therefore, we conclude that the response time of graphene and ITO IPS prototypes is different by no more than the accuracy of the experimental measurements.The turn-off switching time of IPS LC device depends on rotational viscosity of the LC Micrographs and photographs of the graphene IPS prototype cell between crossed polarizers are shown on Fig. Inactive electrodes of graphene IPS pattern illustrate practical challenge for processing of graphene into a high-resolution electrode patterns. Narrow and long electrode lines made from graphene are more susceptible to mechanical damage during photolithography and other processing compared to more robust ITO. Current results demonstrate a successful fabrication of a high-resolution millimeter-scale IPS pattern out of monolayer graphene which is able to sustain processing required for LC devices. However, further optimization of fabrication process is required to improve the yield of photolithography and defect free alignment.In conclusion, we have demonstrated graphene-based single pixel laboratory prototype of an IPS LC device. Photolithographic patterning of single layer graphene into a high-density electrode pattern and a high-quality photoalignment allow efficient electro-optical switching of the liquid crystal. The static voltage response curve and response time of the fabricated graphene-based single pixel laboratory IPS prototype is confirmed to be equivalent to a reference ITO IPS prototype of the same design. Identical electro-optic performance and better optical transmittance of graphene compared to ITO make our findings useful for development of patterned graphene electrodes with a complex shape for displays and other liquid crystal based electro-optical devices on rigid and potentially flexible substrates.4 (~50 Sccm) and H2 (~20 Sccm) as precursor. To transfer graphene, a thin layer of PMMA  was spin coated on Cu foil at 2000 RPM for 1 min. as a carrier layer. The backside graphene was etched by O2 plasma . Then the film (PMMA/Graphene/Cu foil) was left in (NH4)2S2O8 (0.1 mol/L) water solution to etch Cu foil, and subsequently washed in DI water several times to clean the surface. PMMA/Graphene film was next scooped onto target quartz substrate. After the film was completely dry and tightly attached onto the substrate, the whole sample was soaked in acetone to dissolve PMMA. As a result, graphene film was transferred onto quartz substrate.Graphene was grown on Cu foil  at 1000 °C in a low pressure (~500 mTorr) CVD furnace with CH–6 Torr) by Ar gas plasma (8 mTorr) with addition of O2 gas (0.44 Sccm). Magnetron power delivered to sputtering target was 500 W. The thickness of deposited ITO layer was ~10 nm and sheet resistance in the range of 125 Ω/□ − 290 Ω/□. Commercial ITO-coated soda-lime glass used in transmittance study has the thickness of ITO layer of ~25 nm and sheet resistance of 80 Ω/□. The thickness of graphene layer verified by the AFM study is not more than 4 nm, and the resistance of the plain graphene film measured through ITO contacts was in the range 4–15 kΩ, which is consistent with other literature9.The ITO layer was deposited on the quartz glass substrates with graphene films by a pulsed DC magnetron sputtering method. Before sputtering graphene substrates were rinsed with deionized water, isopropanol , and dried in air oven at 90 °C during 10 min. The reactive ITO sputtering was performed in a vacuum chamber  in Oxford 80Plus etching machine. Substrates patterned by both etching steps were stripped from photoresist by rinsing with acetone , and then were rinsed with methanol , dried in air oven at 90 °C during 10 min. Both graphene and ITO substrates were coated with a commercial photoalignment material LIA-01 (DIC Corporation). Alignment layer was deposited by spin coating from a solution with ~2 wt.% of solid material , and dried on a hot plate 85 °C during 3 min. The IPS prototypes cell have cell gap thickness of 3.5+/− 0.2 µm controlled by the diameter of particle spacers dispersed in an optical adhesive  glue seal. Assembled IPS prototype cells were exposed to linearly polarized UV light  at a room temperature (21 °C) to generate a high-quality unidirectional planar alignment of LC molecules on the surface of both substrates. The LC is aligned normally to the polarization direction of the UV light and at 15° with respect to direction of patterned IPS electrodes.The ITO sputtered substrates were cleaned for a second time with isopropanol , and dried in air oven at 90 °C during 10 min. Both wet and dry photolithography steps were performed with a positive photoresist S1818 . The substrates for IPS prototypes were spin coated with a diluted photoresist solution  and dried on a hot plate at 95 °C during 1 min. Photoresist coated substrates with graphene and supplementary ITO layers were exposed to a UV light through a Chromium shadow mask and developed in a 2.5% water solution of tetramethylammonium hydroxide. Opened areas of photoresist pattern were exposed to an etching acid solution in order to etch the ITO layer, and later dry etched with O


Drosophila system where we have engineered the genome to replace the endogenous TDP-43 orthologue with wild type or mutant human TDP-43(hTDP-43). In contrast to other models, these flies express both mutant and wild type hTDP-43 at similar levels to those of the endogenous gene and importantly, no age-related TDP-43 accumulation observed among all the transgenic fly lines. Immunoprecipitation of TDP-43 showed that flies with hTDP-43 mutations had increased levels of ubiquitination and phosphorylation of the hTDP-43 protein. Furthermore, histologically, flies expressing hTDP-43 M337V showed global, robust neuronal staining for phospho-TDP. All three lines: wild type hTDP-43, -G294A and -M337V were homozygous viable, with no defects in development, life span or behaviors observed. The primary behavioral defect was that flies expressing either hTDP-43 G294A or M337V showed a faster decline with age in negative geotaxis. Together, these observations implied that neurons could handle these TDP-43 mutations by phosphorylation- and ubiquitin-dependent proteasome systems, even in a background without the wild type TDP-43. Our findings suggest that these two specific TDP-43 mutations are not inherently toxic, but may require additional environmental or genetic factors to affect longevity or survival.Mutations in TDP-43 are associated with proteinaceous inclusions in neurons and are believed to be causative in neurodegenerative diseases such as frontotemporal dementia or amyotrophic lateral sclerosis. Here we describe a  Cytoplasmic protein aggregates are a common pathological feature of many neurodegenerative diseases . The TARTDP-43 is a member of the heterogeneous nuclear ribonucleoprotein family , is exprtbph gene. No significant phenotypes were observed in flies expressing wild type hTDP-43 compared to control flies, suggesting hTDP-43 can fully replace the functions of the Drosophila gene. Also in contrast to TDP-43 over-expression models, the fly lines carrying ALS-associated mutations were viable and only exhibited mild behavioral phenotypes. These results suggest that these two TDP-43 mutations, G294A and M337V, are not inherently toxic, but might require additional genetic and/or environmental factors to exhibit disease-like phenotypes.Using CRISPR/Cas9 genome editing techniques, we generated three fly lines where the coding region of the endogenous TDP-43 orthologue (TBPH) was replaced with either wild type human TDP-43 or one of two mutations; hTDP-43 G294A (identified as a sporadic mutation) and hTDP-43 M337V . In contrast to other studies, in which mutated TDP-43 is expressed in addition to the endogenous TDP-43, these three fly lines express wild type or mutant hTDP-43 in the absence of expression of the fly Drosophila stocks were reared at 25°C using standard procedures [http://flystocks.bio.indiana.edu/): y[1] M{vas-Cas9.RFP-}ZH-2A w[1118] was the parental line used for CRISPR/Cas9 injections and y[1] w[67c23] P{y[+mDint2] = Crey}1b; sna[Sco]/CyO carrying the Cre recombinase was used to remove the DsRed cassette in the transgenic flies. The hTDP-43 transgenic flies and the parental flies were crossed with w1118 at least five times for minimizing the nonspecific effects caused by CRISPR/Cas9  as described previously [Negative geotaxis assays were performed by placing flies (ranged from 1~28 days-old of age) in separate vials , one day before the assays. Flies were transferred to adapted 25 ml tubes to be used as vertical climbing columns. Flies were placed in a 25°C incubator for recovery for 30 min before the experiment and then tapped to the bottom of the tubes and allowed to climb for 60 s and a digital image captured of the entire tube. The whole climbing tubes was captured as an image. The number of flies in the top one tenth  and aged (~ 28 days post eclosion) flies. For both young and aged flies, these results clearly show that there was no difference in the levels of TBPH/hTDP-43 transcript between control flies (expressing TBPH) and flies expressing wild type hTDP-43. By contrast, both mutant lines had slightly reduced transcript levels in young flies , howeverTo assess the levels of hTDP-43 protein expressed in these lines we separated the total CNS proteins of all fly lines into two fractions, Tris-soluble and SDS-soluble fractions, and then analyzed these fractions on immuno-blots using an antiserum against hTDP-43. The Tris-soluble fraction revealed a single immunoreactive band at the expected size for hTDP-43 in the three lines expressing hTDP-43 . No immuTo characterize the molecular phenotypes further, we used antiserum specific to phosphorylated TDP (pTDP) and examined immunoblots of head lysates of the fly lines. In addition, we used an ubiquitination linkage-specific K48 antibody to examine immunoprecipitated protein, pulled-down by a TDP-43 antibody. When the lysates were blotted with the pTDP-43 antibody, a band of~53 kDa was visible in all three of the transgenic fly lines and notable was increased in flies expressing hTDP-43 M337V . When thWe also examined the expression of hTDP-43 in neurons in the adult CNS using immuno-fluorescence to localize the FLAG-tag, fused at the N-terminus of hTDP-43 . These rDrosophila fragile X mental retardation 1 (FMR1), a component of stress granules in Drosophila [The punctate nature of the cytoplasmic TDP-43 in the flies expressing mutant hTDP-43 was reminiscent of stress granules. To determine whether the observed cytoplasmic puncta shared characteristics of stress granules, we co-labeled the CNS using anti-FLAG antiserum as well as an antiserum against osophila –52. AlthNeurodegenerative diseases that include TDP-43 proteinopathies are usually characterized with phosphorylated TDP-43 and ubiquitin positive inclusions in the cytoplasm or the nuclei of neurons . OccasioAll three of the fly lines were viable as homozygotes and to determine whether there were any defects in development, we measured the length of time between hatching and adult emergence (eclosion). No significant differences were observed between control flies and those expressing wild type hTDP-43 and no delays in development were observed with flies expressing either mutant form of hTDP-43 , indicatrd instar larva crawled during a 5 minute observation period. No differences were detected between any of the lines  expressing mutant hTDP-43, however, there was no difference in transcript levels among all the older flies (4 week-old), suggesting that the mutant TDP-43 does not induce the overexpression of the gene itself. We also detected an increase in the levels of soluble hTDP-43 protein in young flies expressing mutant hTDP-43, but these differences were diminished in aged flies, as did the levels of wild-type hTDP-43. It is interesting that in young flies we detected reciprocal changes in transcript and protein levels of hTDP-43. The levels of TDP-43 protein are known to be autoregulated by binding of the TDP-43 protein to the 3’UTR of the TDP-43 transcript [tbph UTR to maintain the transcript-protein homeostasis seen in cell- and mammal models. If the mutant protein has a slower turnover rate compared to wild type protein it would result in slightly decreased transcripts in the mutant fly lines as we have observed. Our observation that hTDP-43 protein levels decrease with age is consistent with previous findings reported from both Drosophila and rodent models [In the current study, we have developed a anscript . We hypot models , 75. Intt models , 76, migt models . In addit models , 78, 79.In contrast to the relatively small differences in the proteins or transcripts among all the fly lines, the proteins immunoprecipitated with TDP-43 serum showed more substantial changes. Although the input levels of TDP-43 were equivalent across the three lines, the levels of TDP-43 immunoprecipitated were highly enriched in samples from the two mutant lines compared to the wild type hTDP-43 line, as assessed by FLAG immunoblots. This result suggests that the epitope of the wild type hTDP-43 is masked, possibly by its partner proteins, since TDP-43 usually functions as a protein complex . This woUnexpectedly, there were only mild behavioral defects associated with expression of mutant hTDP-43 in our lines. The lack of any differences between the flies expressing wild type hTDP-43 and control flies expressing TBPH suggests that hTDP-43 can fully fulfill all of the essential functions of TBPH. However, we expected to observe defects in flies expressing mutant hTDP-43. Development and longevity were not diminished and only minor defects in fecundity and negative geotaxis were observed. We did observe slightly increased longevity in the males expressing hTDP-43 M337V, although we do not know whether this is due to the presence of the mutant protein or as a result of differences in the genetic background of the lines, despite outcrossing all the transgenic lines with the control line. These results, nevertheless, stand in marked contrast to results from over-expression of mutant hTDP-43 in a variety of systems where it has been shown to be highly toxic , 81–85. In summary, we have described the preliminary characterization of fly lines designed to allow the examination of the effects of TDP-43 mutations in the absence of endogenous TBPH. Flies that express mutant forms of hTDP-43 display only mild behavioral and developmental phenotypes yet show a number of cellular and molecular characteristics that appear to parallel the histopathological hallmarks of human neurodegenerative disease. These flies will be invaluable tools to identify interacting genes and the downstream components resulting from TDP-43 dysfunction. Interestingly, a recent report describes using similar CRSIPR/Cas9 technology to introduce point mutations into the TDP-43 orthologue in zebra fish, but at this time no phenotypes were reported .S1 FigAdult fly heads from each genotype were homogenized in Tris buffer see , centrif(TIF)Click here for additional data file.S2 FigA. Wild type hTDP-43. FMR1 positive cytoplasmic puncta are clearly visible (A’). B. hTDP-43 M337V. Both hTDP-43 positive puncta (B) and FMR1 positive cytoplasmic puncta are clearly visible (B’) but no overlap of the two types of puncta were observed (B”). Arrows indicate the hTDP-43 containing puncta in flies expressing hTDP-43 M337V (B) and arrowheads indicate the FMR1-containing puncta. Scale bar is 2μm.Adult brains were fixed and stained with an anti-FLAG (green) and anti-FMR1(magenta) antiserum to label FLAG-hTDP-43  and FMR1 . (TIF)Click here for additional data file.S3 FigA. Only low levels of ubiquitin staining was visible in flies expressing wild type hTDP-43. B&C. Flies expressing mutant hTDP-43 showed sporadic ubiquitin nuclear inclusions. Representative images from flies expressing hTDP-43 G294A (B) and hTDP-43 M337V (C) showing ubiquitin positive puncta overlapping with DAPI staining (arrows). In flies expressing hTDP-43 M337V, ubiquitin-positive puncta were also present in region not labeled with DAPI (boxed area in C”). These preparations were also stained with anti-FLAG antiserum (arrow heads in D). Examination of the merged images (D”) for both ubiquitin (arrows in D’) showed little overlap of TDP-43 and ubiquitin in these preparations. Scale bar is 2μm.Adult brains of flies expressing wild type (A) and mutant hTDP-43 (B&C) were fixed and stained with an anti-ubiquitin (gray) antiserum to label ubiquitin  and DAPI to label nuclei . Merged images are shown in the third column. (TIF)Click here for additional data file.


Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. Over the last decades, sequencing technologies have transformed our ability to investigate the composition and functional capacity of microbial communities. Even so, critical questions remain about these complex systems that cannot be addressed by the bulk, community-averaged data typically provided by sequencing methods. In this Perspective, I propose that future advances in microbiome research will emerge from considering “the lives of microbes”: we need to create methods to explicitly interrogate how microbes exist and interact in native-setting-like microenvironments. This approach includes developing approaches that expose the phenotypic heterogeneity of microbes; exploring the effects of coculture cues on cellular differentiation and metabolite production; and designing visualization systems that capture features of native microbial environments while permitting the nondestructive observation of microbial interactions over space and time with single-cell resolution. This is an exciting time for those of us interested in microbial communities. The technologies available to us now (many hardly imaginable even a decade ago) are allowing new scientific questions to be asked and driving the field forward. For instance, sequencing technologies have provided us with fantastic insights into which microbes are present in specific environments as well as into what their transcriptional states are. However, as important as sequencing approaches remain, these methods typically provide only broad snapshots of the bulk behavior of microbial communities. Developing a deeper appreciation of the factors driving the establishment and stability of microbial communities, as well as a predictive framework to enable their rational perturbation, will require a better understanding of the causal relationships between microbial community members. As I illustrate here, a productive approach to do so is to consider “the lives of microbes.” In other words, we need to develop methods that capture the native features of the environments that these tiny organisms experience in the natural world and that allow us to explicitly interrogate how microbes exist and interact with one another at the appropriate spatial scale: the microscale.Bacillus subtilis  and are developing single-cell imaging systems to address the question of how cells physically partition themselves within microbial communities .In their native environments, microbes most frequently live as members of complex multispecies communities. They are therefore surrounded not only by the neighboring cells that are physically present but also by the metabolic signals that these microbes have secreted. These secreted metabolites are primary drivers of microbial community interactions . More exBacillus cereus, has two structurally distinct activities: killing and biofilm induction , which aims to promote a community of similarly minded researchers who are interested in creating controlled model ecosystems to study microbial communities in response to perturbations.In “thinking like a microbe,” we must remember that microbes typically exist at significantly lower densities and overall cell numbers than are usually examined in laboratory culture. Thus, we must establish methods to investigate populations of cells interacting at the spatial scale at which microbes natively exist: the microscale. Ideally, these study systems would capture some of the physical and chemical complexity of natural systems while still allowing the nondestructive visualization of the spatiotemporal dynamics of individual cells over time. Since my laboratory is focused on soil microbial communities, we are creating such native-setting-like microcosms using an optically transparent soil-like substrate ; in someIn addition to the areas outlined above, technological advances in several areas would dramatically advance our understanding of microbial systems. Our ability to understand microbial interactions would be revolutionized by the ability to detect and identify “wild” and genetically intractable microbes, for instance, by using their distinct autofluorescence signatures. Although this would require significant advances in our optical detection capabilities, it would also substantially increase our ability to examine natural, nonmodel microbial systems. Other revolutionary advances will likely result from our improved ability to combine multiple imaging modalities  to simultaneously detect the spatiotemporal distributions of microbes and their metabolites at the microscale level within microcosms or natural environments. Such methods would allow us to dissect not only which cells are producing metabolites within microbial communities but also which microbes are altering their physiology and metabolism in response to these cues.


Implementation fidelity refers to the extent to which a proposed intervention is enacted as designed and is necessary to determine how much the intervention in question is the primary mechanism in any changes observed. Start2quit was a randomised controlled trial that aimed to improve attendance at the English Stop Smoking Service (SSS). The complex intervention combining computer-tailored personal risk letters and no-commitment (“taster”) sessions aimed at encouraging attendance at the SSS doubled attendance at the SSS and significantly increased abstinence rates, although attendance and abstinence varied between participating SSSs. Assessment of the fidelity of the delivery of the taster sessions to the protocol was embedded into the trial and is the focus of this study.Eighteen SSSs participated in the study. Taster sessions were delivered by SSS advisors in the area. Of the 131 sessions delivered, 93 (71 %) were recorded and 41 (31.3 %) were selected for transcription and analysis. The taster session protocol contained 73 specified behaviours, which were independently classified into component behaviour change techniques (BCTs) using an established taxonomy for smoking cessation. All transcripts were coded by two authors with 25 % additionally coded by a third. The fidelity of each taster session was expressed as the percentage of overall protocol-specified behaviours that were delivered. Adherence to each BCT was measured as the number of behaviours applied by the advisors within each BCT divided by the total number classified within each.Adherence of protocol-specified behaviours was relatively high (median 71.23 %), though there was considerable variation (28.76 to 95.89 %) in individual sessions. Median fidelity to specific BCTs across sessions also varied from 50 to 100 %. Shorter sessions, sessions run jointly by two advisors, by female advisors, or by advisors aged 45 to 54 were associated with higher levels of adherence. There was no association between adherence and subsequent attendance at the SSS.These results suggest that the delivery of the intervention of this study is not likely to have been impacted by issues of fidelity. As such, we can have greater confidence that variability in the main outcome is not due to variability in SSS advisor adherence to the protocol of the taster sessions.ISRCTN76561916Current Controlled Trials  Implementation fidelity refers to the extent to which a proposed intervention is enacted as designed . Three cThe assessment of treatment receipt and treatment enactment is a complex process involving the verification of participant understanding of intervention content and the monitoring of participant use of treatment-related strategies in everyday life . MeasureDespite this, a systematic review of psychosocial treatments by Perepletchikova and colleagues  found thRecent research has begun to address this issue and to propose methods to monitor the fidelity of delivery within study interventions . In partIn addition to identifying the extent of intervention delivery fidelity, it is also important to understand the ‘essential’ components of an intervention , 12. BehIn this paper, we report on an assessment of the fidelity to the protocol of one part of a complex intervention of a randomised controlled trial. The Start2quit trial  aimed top < 0.001). Seven-day point-prevalent abstinence at the 6-month follow-up, validated by salivary cotinine was also significantly higher in the intervention group . The number completing the 6-week SSS course was also significantly higher in the intervention than that in the control group . However, some variation was found in both attendance and in 7-day validated abstinence between SSSs. Overall attendance between SSSs varied from 2.1 to 23.1 % and validated 7-day abstinence from 2.1 to 13.4 % [Participants of the Start2quit trial randomised to the intervention group received a computer-tailored personal risk letter and an invitation to attend a no-commitment ‘taster session’ to find out more about the support provided by the SSS. These taster sessions were delivered by trained SSS advisors and were based on a detailed treatment manual. Results from the trial showed that this intervention more than doubled the odds of attendance at the SSS during the 6-month period between randomisation and follow-up as measured by SSS records, compared to the control group who received a standard generic invitation to contact the service  . This stn = 106,819) were sent an invitation to participate in the study along with a Smoking Behaviour Questionnaire (SBQ). Smokers returning the questionnaire and giving consent (n = 4384) were randomly allocated to the control group (n = 1748) or to the intervention group (n = 2636). Those in the control group were sent a generic letter advertising the SSS in their area and those in the intervention group were sent a computer-tailored letter signed by their GP containing personalised risk information and an invitation to a no-commitment ‘taster session’ to find out more about the SSS.Eighteen SSSs across England and 99 general practices within the SSS areas were recruited into the trial between February 2011 and October 2013. All current cigarette and roll-up smokers over the age of 16 identified in participating practices . If there was more than one recorded session available for a lead advisor, one session was selected at random.Of the 131 sessions delivered, 93 were recorded (71 %). The remainder were not recorded due to forgotten recording equipment, equipment failure or no consent for recording by one or more participants attending the session. Due to the quantity and length of the recordings, sessions to be transcribed and analysed were purposively selected to ensure one session by each lead advisor (where available) was included .n = 222).Attendees in this study were defined as those participants of Start2quit who were randomised to receive the intervention and who also attended a taster session included in this analysis  or instructions that they should follow (e.g. ask attendees how many of them enjoy smoking). A coding frame was developed by the first author based on this protocol. Two additional researchers verified the frame by checking that it contained all behaviours specified in the taster session protocol.The behaviours specified in the coding frame were independently classified by two of the authors into component BCTs using an established taxonomy of 45 smoking cessation BCTs . This waTaster session characteristics included the structure of the session (one advisor or two providing content), the length of the session and number of attendees.All advisors were recruited on the basis that they were employed by the SSS and had some previous experience of assisting people to quit smoking. Advisors also completed a short questionnaire at the time of training. Data gathered included gender, age, highest educational qualification, type of smoking cessation training, time since smoking cessation training, employer, job title and number of patients seen in the previous 6 months.The proportion of attendees at each session who subsequently attended the SSS, and the proportion of attendees at each session who were found to be 7-day point-prevalent abstinent at the 6-month follow-up, biochemically validated by salivary cotinine, were measured. It is important to note that attendance at the SSS was the behaviour targeted by the taster sessions and the one hypothesised to be related to the fidelity of delivery of the intervention. However, 7-day point-prevalent abstinence data was also included to test the hypothesis that there was no link between this outcome and adherence given the potentially complex pathway between the taster sessions and cessation.All transcripts were anonymised for SSS area and advisor. Each transcript was coded by a minimum of two authors; specifically MSM coded all transcripts and DK and AH coded 50 % each. The remaining authors (HG and SG) coded 25 % of the transcripts between them, chosen at random, to provide an extra level of reliability checking. Average inter-rater reliability for coding was 86 % (68–99 %) across sessions. All disagreements were resolved through discussion between researchers. Data from the coding frames were double entered into Excel and discrepancies corrected.The fidelity of each taster session was expressed as the percentage of overall protocol-specified behaviours that were delivered, that is the number of protocol specified behaviours applied by the advisor divided by the total number of behaviours . Adherence to each BCT was measured as the number of behaviours applied by the advisors within each BCT divided by the total number classified within each.t tests and analysis of variance were used to assess differences in adherence to the protocol by advisor characteristics and advising structure. Correlations were computed to explore the association between adherence to protocol-specified behaviours and the length of session. To assess whether adherence was related to the main outcome measures of attendance at the SSS and 7-day point-prevalent abstinence, high and low adherers were split into two groups (high adherers were those sessions that reached the median and above), and t tests used to assess differences in outcomes between these groups. Data were analysed in SPSS (v22).Mean and median adherence to protocol specified behaviours and for each BCT was calculated. The study was approved by the by the South West London Research Ethics Committee, and R & D approval was obtained from all participating SSSs.Mean session duration was 43 min and 4 s and ranged from 14 min to 1 h and 18 min. Twenty-seven sessions (65.85 %) were facilitated by one lead advisor with minimal administrative support by an additional advisor, and 14 (34.15 %) were split between two advisors with one taking the lead. The number of smokers attending a session ranged from 1 to 17 .Only lead advisor characteristics were assessed due to their leading role in the taster sessions and the significantly greater proportion of the information that they communicated to attendees. The majority of advisors were female (73.17 %) and 51.22 % were aged between 45 and 54. The majority were educated to degree level or higher (73.17 %), 41.46 % had received smoking cessation training to NCSCT  stages 1 and 2 with various additional training and 60.98 % had received training within the previous 3 years. All were employed by the SSS and 70.73 % were employed as SSS advisors. Most (90.24 %) had seen 21 or more patients in the last six months (Table m = 68.53 %) of protocol-specified behaviours was delivered across sessions, though there was considerable variation. Adherence in individual sessions varied from 28.76 to 95.89 %  to 100 % in five of the BCTs Fig. . Howeverp = 0.044). There was a negative correlation between session length and adherence, thus shorter session length was associated with increased adherence to protocol-specified content .Sessions where the advisor was assisted by a second advisor were found to have significantly higher adherence levels than sessions run by one advisor alone . Level of adherence also varied with advisors’ age, those aged between 45 and 54 were significantly more adherent (75.41 %) than advisors who were younger (61.87 %) or older (60.45 %) (p = 0.021). Full details including CIs are reported in Table Female advisors had significantly higher levels of adherence to protocol-specified behaviours than male advisors , and the mean proportion of attendees who were found to be validated 7-day point-prevalent abstinent at the 6-month follow-up was 20.21 % (range 0 to 66.67 %).The proportion of attendees who subsequently attended the SSS ranged from 0 to 100 %  or in the proportion who were 7-day point-prevalent abstinent .This study represents a thorough assessment of intervention delivery fidelity to the taster session protocol of the Start2quit trial. The median adherence to specified behaviours of 71.23 % was high relative to similar evaluations , 22. HowThis study has some important implications for future fidelity assessments of novel interventions. Firstly, the finding that more specific techniques were more likely to be delivered suggests the importance of considering the previous training and expertise of those delivering an intervention. While it has been reported elsewhere that more specific BCTs are less likely to be delivered reliably , this waThe finding that sessions run by two advisors had higher levels of adherence than those run by a single advisor has potentially significant implications for future implementation assessments. One possible explanation for this result is that the second advisor acted as a safety net, delivering key items that the first advisor failed to present. Research in the field of sociology has suggested that co-facilitation of training sessions can be beneficial due to shared responsibility and mutual support , 25. ThuThis study also examined specific advisor characteristics. Being female and being aged between 45 and 54 were significantly associated with adherence to the protocol. These findings should be treated with caution due to the small sample size, the fact that not all advisors were included in the analysed sessions, and the omission from the analysis of the contribution of secondary advisors who delivered a minority of the content. However, they do indicate a need for more research into how advisor characteristics potentially influence adherence to study manuals and how this influence can be mediated through training. To date, the exploration of the reasons for variation in adherence has been largely ignored with the exception of some work on the impact of practical issues such as lack of time .While the median adherence to specified behaviours was high, there was considerable variability across study areas suggesting some modifications be made to the training provided to advisors if this intervention were to be adopted. Specifically, it may be beneficial to tailor training to advisor experience levels in delivering behaviour change interventions. In addition, given that the two BCTs with lowest adherence were related to providing information on the SSS itself, greater emphasis on the importance of communicating this practical information may be needed.However, the lack of association between adherence to BCTs and the outcomes of attending the SSS and 7-day abstinence suggests that absolute fidelity may not be necessary for this intervention to be effective. Absolute fidelity is rarely realistic in real-world settings, and in this intervention, it is important that advisors have some freedom to adapt to the specific needs of the people to whom they are presenting. As discussed by Dusenbury et al. , key to Finally, the lack of association between adherence and study outcomes may have been influenced by the generally high levels of motivation of attendees. Responding to an invitation to a smoking cessation study and then attending a session to find out more about the SSS indicates a high level of interest in both quitting smoking and availing of SSS support to do so. Perhaps this enthusiasm meant that the presentation of protocol-specified content was less important than theorised as attendees were already inclined to sign up for an SSS course. In addition, receiving the first half of the intervention, a personalised risk letter, may have meant that the likelihood of participants changing their behaviour had already increased and as such they were more amenable to attending the SSS or quitting smoking.A key strength of this study is the robust methodology. All sessions were double-coded and 25 % were triple-coded. Sessions were anonymised for area and advisor to remove potential bias and inter-rater agreement was high for all sessions. The protocols were highly specific and all advisors underwent a 2-h training session. In addition, as previously mentioned, all advisors were experienced in delivering smoking-related behaviour change interventions.A significant limitation of the study is its evaluation of only a sample of the taster sessions delivered as part of the Start2quit study, due to the prohibitive resources required to transcribe and analyse the additional 47 sessions recorded. However, the 41 sessions analysed represent 31.3 % of the total session number, well over the 20 % minimum recommended by Schlosser , 28. OthWe did not assess the other key components of implementation fidelity, intervention receipt and enactment. These additional measurements were outside the scope of this study but could have provided clarity on the efficacy of specific BCTs. Finally, the high rate of unrecorded sessions is an issue and it is possible that when this was due to tape failure, it was not random. For example, less well-organised and prepared advisors may have been less likely to remember to record and operate the machine correctly.Assessing implementation fidelity is essential in ensuring that research studies are actually measuring that which they claim to be measuring , 7. This


In recent years, the demand for high data rate wireless communications has increased dramatically, which requires larger bandwidth to sustain multi-user accessibility and quality of services. This can be achieved at millimeter wave frequencies. Graphene is a promising material for the development of millimeter-wave electronics because of its outstanding electron transport properties. Up to now, due to the lack of high quality material and process technology, the operating frequency of demonstrated circuits has been far below the potential of graphene. Here, we present monolithic integrated circuits based on epitaxial graphene operating at unprecedented high frequencies (80–100 GHz). The demonstrated circuits are capable of encoding/decoding of multi-gigabit-per-second information into/from the amplitude or phase of the carrier signal. The developed fabrication process is scalable to large wafer sizes. To meet the fast growing demand for telecommunication services, developing high-data-rate communication links in the range of multi-gigabit per second (Gbps) is necessary. The high speed data links can be implemented using either wireless or fiber optic technologies. Wireless technology, particularly in urban areas, has several advantages over fiber optics such as mobility, universal deployment, short installation time and cost effectiveness. However, to achieve data rates comparable to that of the fiber optics, there is a need to develop wireless systems with a very large bandwidth ∼10 GHz). This may be achieved by operating at millimeter wave (mm-wave) frequencies (30–300 GHz)0 GHz. ThT) of 400 GHz and maximum oscillation frequency (fMAX) of 100 GHz have been demonstrated3567691011131415171819212223In this regard, graphene is a promising material for the development of mm-wave electronics due to its excellent electron transport properties. Recently, there is a rapid progress in the development of graphene field effect transistors (G-FETs). G-FETs with intrinsic current-gain cutoff frequencies  based on epitaxial graphene in high data rate applications in the 90 GHz band. The developed process is scalable up to full wafer sizes. Currently the limiting factor is not the wafer size but the uniformity of available epitaxial graphene resulting in about 70% yield. The fabricated MMIC has different circuits elements capable of receiving and retrieving information embedded in the amplitude and phase of the carrier signal at the rate of 4 Gbps. Furthermore, the developed circuits are highly linear allowing to generate modulated signal up to the rate of 8 Gbps at 90 GHz band with a bit-error-rate (BER) below 102/V and 150–250 Ω/Vs respectively. More details of graphene growth and its properties can be found in the method and Typically, the term “epitaxial graphene” refers to graphene grown on SiC which is in fact not done by epitaxy but by sublimation of silicon. In this study graphene is grown by traditional Chemical Vapor Deposition (CVD) epitaxy using carbon precursor or more accurately by Vapor Phase Epitaxy (VPE)To achieve complete wireless communication systems based on graphene it is needed to realize all the above mentioned components with graphene technology. Transistors are versatile elements and by choosing proper operating points and passive components, all needed functionalities are attainable.Therefore, G-FETs are the only needed graphene based active elements for these circuits. In addition, it is desirable to have whole transmitter and receiver chain on a single chip which facilitates the very broadband performance needed for the high data rate applications. Hence, developing G-FET based MMICs is a key requirement to obtain graphene based high speed wireless systems.MAX more than 200 GHz is needed. Therefore, more development in G-FET technology is needed to realize G-FET based amplifiers at this frequency band. However, for other applications where the G-FETs operate in other modes, the existing G-FETs technology based on hydrogen intercalated graphene is sufficient. This is because this type of graphene exhibits a high carrier mobility with a very low sheet resistance. It results in a reduction of channel resistance and consequently achieving small time constants in G-FETs needed for higher frequency operations. Developing microstrip MMIC process on SiC substrates is a laborious task especially at high frequencies where the parasitic elements and unwanted propagation modes can significantly deteriorate the circuit performance. In addition, graphene compatibility is extremely important and because of that many existing circuit fabrication technologies cannot be directly applied for the development of graphene based MMICs. These process have several steps that can potentially degrade graphene. One way to avoid this problem is to fabricated the passive elements first and then transfer graphene and perform G-FETs fabrication2O3 deposited by the atomic layer deposition (ALD) method2O3 exhibits a poor thermal conductance and a high tensile stress. Since SiC via hole etching is performed at very high powers, the generated heat causes the Al2O3 layer to crack. This completely damages G-FETs as well. To avoid it, a thin layer (15 nm) of ALD Al2O3 is deposited and it is followed by 100 nm SiN sputtered at a very low density plasma. This process does not damage the graphene. In addition, the new encapsulated layer can completely protect graphene from the remaining plasma steps. Furthermore, the new layer has a better thermal conductance and a lower stress and can survive the via hole etching process. In this process the SiC substrate is thinned down to 70-μm providing a platform to develop circuits up to 250 GHz. By using our device and circuit technologies we are able to realize needed RF modules except amplifiers at 90 GHz band. RF signal in mm-wave can be generated by using G-FET based frequency multipliers. However, since the output power of these frequency multipliers are very low67Circuit performance depends on both device and circuit technology. Device (G-FET) technology has recently shown a rapid progress. However, to have G-FET based amplifiers operating at 90 GHz band, G-FETs with fn layer 1 nm of ALThe most fundamental digital modulation techniques are binary amplitude-shift keying (BASK) and binary phase-shift keying (BPSK) where binary information is coded in the amplitude and phase of the carrier signal respectively . A mixerTo evaluate the performance of the power detector based demodulator MMIC, a BASK signal at 90 GHz with modulation rates of 1, 2 and 4 Gbps is used. Due to the band width limitation of our arbitrary waveform generator, the BASK signals have triangular-shaped pulses . The eyeThe performance of the demonstrated G-FET based demodulators and this work is shown in −6 can be estimated. The experiment is repeated by 16-QAM signals modulated at the rate of 4, 8 and 16 Gbps. The reconstructed constellation diagrams are plotted in −6, 10−5 and 10−4 respectively. It can be seen that the achieved constellation diagrams are not distorted meaning that the mixer is highly linear. The MMIC mixer circuit can be used for signal modulation as well. As shown in 29To achieve high frequency devices base on graphene having a high carrier mobility is not sufficient. Graphene films should have high carrier density as well in order to obtain low sheet resistance levels needed for high frequency applications. These properties exist in hydrogen intercalated epitaxial graphene and thereby it is more beneficial for RF devices and circuits. The development of G-FET MMICs that can perform multi-Gbps signal modulation and demodulation at mm-wave makes a start on the realization of complete high data rate transceivers based on graphene. Prior to this work, the operating frequency and data rate of the demonstrated graphene based ICs are far below the performance of the existing technologies. This is mainly due to the lack of high quality process technology and material. For high data rate communication, the linearity of the RF components plays a key role. The fabricated MMIC mixer is highly linear allowing to generate and retrieve multi-Gbps data at 90 GHz atmospheric window. This work demonstrates G-FETs’ great potential for high data rate communication systems. To achieve complete wireless communication systems based on graphene, more development in G-FET technology is needed to realize amplifiers at mm-wave band.2 nominally on-axis 4H-SiC (0001) or 6H-SiC (0001), Si-face semi-insulating chemo-mechanically polished substrates. Graphene layers are grown under an argon laminar flow at hot-wall Aixtron VP508 and Aixtron G5 reactors where graphene growth on 4″ SiC substrates is under development. The process depends critically on the creation of the flow conditions in the reactor that control Si sublimation rate and enable mass transport of hydrocarbon to the SiC surface. Reynolds number (Re) measures the ratio of inertial forces to viscous forces and consequently quantifies the relative importance of these two forces in a given gas flow. Tuning the value of the Re number allows to form a thick enough Ar boundary layer to prevent Si sublimation. This lets the diffusion of hydrocarbon to the SiC surface and growth of epitaxial graphene. The intercalation of hydrogen is obtained in situ at the temperature of 1100 °C and 900 mbar pressure. In our samples the presence of hydrogen atoms is maintained up to 700 °C, high enough to meet the requirements of high-speed electronics and high-temperature sensing.Epitaxial graphene is grown at 1600 °C and 30 mbar pressure by CV technique on (15 × 15) mm2O3 (15 nm) and sputtered SiN (100 nm) layers are utilized. The rest of the fabrication process are based on photolithography. For forming thin film resistors, tantalum Nitride (TaN) is sputtered. The metal-insulator-metal (MIM) capacitors are based on SiN. After the fabrication of MIM capacitors, dielectric via etching is performed to reach to the G-FET metal pads for the deposition of the large pads and transmission lines. Gold electroplating is done to reduce the metal resistivity and form air-bridges. Then a thick layer of resist is used to protect the surface and the chip is glowed to a carrier wafer for the back side processing. The substrate is thinned down to 70 μm and polished by a CMP process. For via hole formation, a thick layer of nickel is plated as a hard mask and high power ICP is used to etch via holes. The nickel mask is wet striped and a seed layer is sputtered on the back side of the chip. Finally, gold is plated for the metallization of the ground plane and via holes.The fabrication process has about 30 different steps including 3 ebeam lithography and 13 photolithography steps. Ebeam-lithography is used for the fabrication of G-FETs since ebeam resists are more compatible with graphene. For G-FET passivation a combination of ALD AlAn Agilent signal generator and an OML S10MS W-band source module are used to provide the LO signal. The digital modulated signals at baseband and intermediate frequency are generated by a Keysight M8195A 65-Gsps sampling rate 8-bit arbitrary waveform generator. The millimeter wave output is measured by a Lecroy Labmaster 10-100Zi real time oscilloscope which has 100 GHz input bandwidth and 240 Gsps sampling rate. High data rate BASK (on off keying) signal is generated by an in-house fabricated W-band frequency multiplier module based on InP DHBT MMIC. The data captured by the real time oscilloscope is processed using Matlab code.How to cite this article: Habibpour, O. et al. Wafer scale millimeter-wave integrated circuits based on epitaxial graphene in high data rate communication. Sci. Rep.7, 41828; doi: 10.1038/srep41828 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The adsorption state was probed by analysing the G-peak position in the measured in-situ Raman spectrum during electrochemical experiments. We conclude that electrochemical Raman spectroscopy on graphene is a valuable tool to obtain in-situ information on adsorbed species on graphene, isolated from the rest of the electrochemical behaviour.Electrochemistry on graphene is of particular interest due to graphene’s high surface area, high electrical conductivity and low interfacial capacitance. Because the graphene Fermi level can be probed by its strong Raman signal, information on the graphene doping can be obtained which in turn can provide information on adsorbed atoms or molecules. For this paper, the adsorption analysis was successfully performed using three electroactive substances with different electrode interaction mechanisms: hexaammineruthenium(III) chloride (RuHex), ferrocenemethanol (FcMeOH) and potassium ferricyanide/potassium ferrocyanide (Fe(CN) Graphene has received much attention during the last decade because of its unique properties such as mechanical strength and impermeability to gasses12341101112141516171819202119232526Here we will exploit the technique of Raman spectroelectrochemistry to obtain information about, among others, the reaction kinetics during Faradaic reactions on the surface of graphene. Often the desorption phase is the rate-determining step in nano- and microfluidic systems, where mass transport by diffusion is relatively fast compared to large scale systems. E.g. in redox cycling experiments slow desorption results in a lower effective diffusion coefficient and thereby in reduced signal levelsIn the pathway of electrochemical reactions several steps can be distinguished as shown in 6 (potassium ferricyanide/potassium ferrocyanide), which is known to have different reaction kinetics on monolayer graphene and pyrolytic graphiteTo explain redox reactions for complex molecules two mechanisms are recognized: inner-sphere and outer-sphere electrode reactions121010Sm will then be a superposition of two spectra: the graphene with adsorbed molecules Sad and the bare graphene Sgr. For the ratio between both we define an occupation factor β as followsWhenever molecules adsorb to the graphene surface, the graphene Fermi level is shifted by local doping (changing the carrier concentration), which can be probed by Raman spectroscopy19when this factor is low β ≈ 0 solely the bare graphene spectrum is measured, indicating no interaction between the graphene and the molecules in solution. For a large factor β ≈ 1 the graphene is entirely occupied by adsorbed molecules which interact with the graphene and therefore change the Raman spectrum.−1) is analysed, since this is the most sensitive indicator for the graphene Fermi level and therefore the adsorption level8CNP , which is the applied gate voltage corresponding to a minimum in the G-peak position curve, probing the graphene Fermi level. This will cause the valley to move horizontally A typical Raman spectrum measured for our graphene is shown in in-situ Raman spectroscopy experiments the adsorption (and possibly the approach) of redox species can be probed independently of the remainder of the electrochemical behaviour. To explain the electrochemical voltammogram data, we will thus distinguish adsorption and electron exchange at reactive sites 6 was measured using the second graphene device.To test our hypotheses, the adsorption of the three redox active species during electrochemical experiments using k0, therefore we conclude that the electrode kinetics of RuHex are such that it is a fast reactionin-situ recorded G-peak position curves are displayed, which are at about the same position as the baseline curve. Both curves are constant over several measurements, showing no change over time. This dataset indicates that RuHex adsorption to the graphene electrode was absent or below our detection limit (β ≈ 0). As expected for an outer-sphere redox couple that does not need adsorption to react, a sizeable redox current was still observed despite this lack of adsorption.The cyclic voltammogram of RuHex at the graphene electrode is shown in k0 . Both effects can be explained by the adsorption of FcMeOH molecules. Furthermore, the valley  than for RuHex (0.10 V) and FcMeOH (0.14 V), indicating a much lower standard rate constant k0 (see The spectroelectrochemistry of Fe(CN)t k0 see  and thus6 adsorption, whereby the difference between the first and the subsequent measurements suggest that two adsorption states exist. In addition the slope becomes shallower. The observations are as expected for the inner-sphere redox couple Fe(CN)6, that needs adsorption before reacting.In 6 can be found, the reservoir was three times rinsed with background electrolyte and subsequently the measurements were performed. The results are shown in 6. The voltammogram shows a quite different behaviour: the peak separation voltage has reduced from approximately 0.9 V to approximately 0.4 V and the current has decreased 50-fold. The decrease in peak separation voltage could be explained by a difference in standard rate constant resulting in different overpotentials. The voltammogram in 6 on graphite, where reaction takes place at reactive edge sites with low overpotential. The traces in 6. In the first measurement the minimum in the G-peak position curve is still quite similar to the position in 296 that is hard to desorb, similar to FcMeOH.To test whether also evidence for desorption of Fe(CN)6 activity was verified in the Fe(CN)6 solution with the results as shown in 6 can be found, showing to what extent the device history influences its behaviour.Finally, a measurement was performed to establish whether the signal shown in β, as visible from the Raman spectrum, and the amount of reacting species were directly coupled. The data for RuHex, an outer sphere redox substance, were not in conflict with this model, as for such substances redox activity is independent of adsorption. For FcMeOH, another outer sphere redox substance, adsorption was still found. This, however, is in accordance with literature reports, whereby it could not be established whether this adsorption contributes to the redox activity. The results for Fe(CN)6 were also in line with expectations, as we observed both adsorption and electroactivity for this inner sphere redox couple. We however observed in the Fe(CN)6 desorption experiments that two type of reactions occur, with low and high overpotential. The latter one has a major contribution to the current. The former one is persistent and remains when the solution is flushed.In the model, the occupation factor CNP shifted to a more negative voltage and for Fe(CN)6 (−3 and −4 charge) to a more positive voltage, suggesting that the charge of the molecule determines whether n- or p-doping is induced. Secondly, graphene electron mobility is reduced by grain boundaries 3739A further complication in the analysis of the cyclic voltammograms is that the graphene conductivity is influenced by adsorbed species in two ways. Firstly, the adsorbed molecules on the basal plane  influence the conduction by changing the carrier concentration. For FcMeOH (uncharged) the charge neutrality point Vin-situ Raman spectrum during electrochemical experiments. Prior to every experiment a G-peak position curve was recorded for the bare graphene, serving as a baseline for subsequent measurements. For RuHex no change in the G-peak position curve was found during the electrochemical experiment compared to the baseline in solely background electrolyte, which was as expected. In case of FcMeOH a clear stable negative shift in the G-peak position curve was observed, indicating a strong adsorption state to the graphene, potentially caused by π-interactions or electrostatic interactions. For Fe(CN)6 a clear positive shift in the G-peak position curve could be seen, however for this species the measurement required some time to settle towards a constant voltammogram and G-peak position curve. This is suggesting that FcMeOH and Fe(CN)6 interact differently with the graphene. Furthermore, when measuring Fe(CN)6 on the graphene electrode after FcMeOH we found no clear difference in the voltammogram compared to Fe(CN)6, while the G-peak position curve indicated that both species were adsorbed.Evidence for the adsorption state during redox reactions at graphene in the Raman spectrum was found. The adsorption state was probed by analysing the G-peak position in the measured in-situ information on adsorbed species on graphene, independent of the cyclic voltammograms measured. This makes graphene an even more interesting electrode material for electrochemical sensing as it allows a fundamental investigation of detailed adsorption mechanisms by Raman spectrometry. We found indications that the adsorption of species for the larger part takes place on the less reactive basal plane, while local reactive sites mainly determine the electrochemical behaviour. We also observed additional effects of the adsorbed species to the graphene surface, where a slight change in the electrochemical behaviour is most probably caused by changes in graphene charge carrier density and passivation of the surface.Based on these findings we conclude that Raman spectroelectrochemistry on graphene is a powerful tool to obtain et al.−10 m2/s. Current is calculated by integrating the normal flux at the electrode boundary over its area and multiplying by F. On t = 0 is the concentration of the reduced species 1 mM and the oxidized species are absent. The transient behaviour is simulated by scanning the overpotential between −1 V and 1 V with 25 mV/s.The peak potential separation as function of the standard rate constant is simulated in COMSOL Multiphysics 5.1 using the ‘Transport of Diluted Species’ physic module. A round electrode is modelled in a cylindrical coordinate system with boundary condition: 2 for both devices.The schematic of the fabrication and measurement setup of the graphene devices are shown in fcutoff = 5 Hz). To the background electrolyte, 0.1 M KNO3, the reactive species 6) were added in a concentration of 1 mM. Subsequently, 100 μL of solution was applied to the graphene device. All potentials were measured against an Ag/AgCl reference electrode (WPI Dri-Ref-450). The spectra are recorded using a WITec alpha 300 system with a 532 nm laser at 1 mW with an immersion objective for measuring in liquid (Nikon Fluor 40x 0.8 W DIC). Analysis of the recorded Raman spectra is performed using a MATLAB script, which accounts for an offset in the spectrometer by fitting the Rayleigh peak. Next Lorentzian curves are fitted to the graphene peaks to obtain their positions and intensities. The voltammetric measurement protocol was as follows. In each measurement three scans were performed in series (whereby the results of the last two scans are displayed). Measurements in a series were performed with a time spacing of approximately 1 minute.The setup used in this experiment as shown schematically in How to cite this article: van den Beld, W. T. E. et al. In-situ Raman spectroscopy to elucidate the influence of adsorption in graphene electrochemistry. Sci. Rep.7, 45080; doi: 10.1038/srep45080 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.