Lymantria dispar L. (Lepidoptera: Erebidae)) is a serious pest of hardwood forests. In the search for an environmentally safe means of its control, we assessed the impact of different concentrations of essential oils (EOs) from the seeds of three Apiaceae plants  on behavior, mortality, molting and nutritional physiology of gypsy moth larvae (GML). EOs efficacy was compared with commercial insecticide NeemAzal®-T/S (neem). The main compounds in the Eos were trans-anethole in anise; carvone, limonene, and α-phellandrene in dill; and trans-anethole and fenchone in fennel seed. At 1% EOs concentration, anise and fennel were better antifeedants and all three EOs were more toxic than neem. Neem was superior in delaying 2nd to 3rd larval molting. In the 4th instar, 0.5%, anise and fennel EOs decreased relative consumption rate more than neem, whereas all three EOs were more effective in reducing growth rate, approximate digestibility and efficiency of conversion of food into body mass leading to higher metabolic costs to GML. Decrease in consumption and metabolic parameters compared to control GML confirmed that adverse effects of the EOs stem from both pre- and post-ingestive mechanisms. The results indicate the potential of three EOs to be used for gypsy moth control.The gypsy moth ( Lymantria dispar L. (Lepidoptera: Erebidae) is a polyphagous insect that feeds on over 500 plant species within 73 families, but the most suitable hosts are oaks (Quercus spp.) [Gypsy moth us spp.) ,2. In Seus spp.)  and overus spp.) ,7. Gypsyus spp.) ,9.The use of conventional insecticides, often in an inappropriate manner, bears the risk of evolution of insect resistance and may lead to severe environmental disturbances due to pollution and adverse effects on non-target organisms ,13,14,15Plant secondary metabolites are diverse chemical compounds synthesized through metabolic pathways derived from primary metabolism . AlthougAthamanta haynaldii (Borb. Et Uecht.) the EOs provoked almost three times lower leaf damage by GML than in the control group [Pimpinella anisum L., dill Anethum graveolens L., and fennel Foeniculum vulgare Mill.) against GML by assessing their insecticidal and antifeeding activity as well as influence on larval molting, growth and food utilization. It was expected to find a new candidate for developing ecofriendly EO-based insecticide against GML.Apiaceae species are rich in essential oils whose insecticidal and repellent activities have been confirmed in many studies on ticks, mosquitoes, cockroaches, stored products, and crop pests ,60,61,62ol group . This stEssential oils (EOs) were isolated from seeds of three Apiaceae species—anise, dill, and fennel. Anise seed EO is composed of twelve compounds , among which the most dominant constituent was phenylpropanoid trans-anethol . Sixteenp = 0.003; 0.1 vs. 1% and 0.5 vs. 1%: p ˂ 0.001). Significant Bot × Conc interaction indicated that GML sensitivity to increasing concentration differed among EOs and neem , concentration and their interaction on variation of absolute deterrence coefficient (ADC) A. It canand neem A. NamelyAntifeeding activity of the EOs was also confirmed in the choice assay. Variation in applied botanical type and in their concentration showed a significant impact on the relative deterrence coefficient (RDC) (ANOVA results in The sum of an absolute and a relative coefficient (Tot coefficient), showed the best antifeeding activity in anise seed oil C. ConcenThe effect of three EOs and a neem standard incorporated into the diet on larval mortality and molting was estimated after 120 h (24 h of exposure to EOs or neem followed by 96 h of feeding on control diet). As revealed by two-way ANOVA A the perLarval molting into the 3rd instar was also significantly affected by the botanical type and its concentrations . Larvae exposed to EOs and neem ate 35–74% less food  and showed 40–82% reduction in the amount of assimilated  and 24–67% reduction in the amount of metabolized food . Differences of treatment groups from the control group were mostly highly significant  and RCR  differed significantly among experimental groups and exposure to 0.1, 0.25 and 0.5% concentration of EOs and neem led to significantly lower RGR (70–119% reduction) and RCR (42–67% reduction) values comparing to the control group . Only 0.1 and 0.25% dill EO and 0.25% fennel EO acted mainly as feeding deterrents  showed negative values and food consumption relative to initial mass (RCR) had the lowest values at 0.5% EO A,B. RGR ol group . Besidesterrents .12,43.642 = 41.37, p < 0.001; ECD: F12,43.45 = 24.97, p < 0.001) leading to 8–34% and 47–184% reduction respectively. However, whereas AD was significantly reduced in the majority of EO treated groups, ECD showed significant change only at the highest EO concentration  which was most probably caused by a significant decrease in the amount of assimilated food. It can be noticed from Decreased ECI can be a consequence of a lower proportion of assimilated relative to consumed food (AD) and/or a lower proportion of assimilated food allocated towards growth (ECD). EOs and neem treatments significantly affected both indices  EOs and neem did not differ in the impact on nutritional indices A, ECI FD, AD FiE, ECD FF, and inThe impact of essential oils on behavior, survival, and reproduction of various pest insects has been widely studied ,64,65. RAthamantha haynaldii and Myristica fragrans Houtt. EOs. At concentration of 0.1% these EOs showed two times higher absolute deterrence coefficient than anise EO. Additionally, Tanacetum vulgare L. EO delayed molting more effectively than Apiaceae EOs in our study [Ocimum basilicum L. and T. vulgare EOs, and oil-in-water EO emulsions from Thymus herba-barona Loisel. and Rosmarinus officinalis L. [Aesculus hyppocastanum L. and Morus alba L. [Anise and dill seed EOs appeared to be the most effective agents against 2nd instar GML since anise EO had good antifeeding activity and both EOs induced high mortality and delayed larval molting. Kostić et al.  found stur study . On the nalis L. ,74,75,76 alba L. . Pseudaletia unipuncta Haworth [Musca domestica L. larvae [Spodoptera littoralis Boisduval [Tribolium castaneum Herbst [Our findings are in accordance with toxic, antifeeding, and molting delay effects of Apiaceae EOs described in other insect species. Anise, dill and fennel EOs had good larvicidal, repellent, and antifeeding effects on lepidopteran pests ,78,79,80 Haworth  and bett. larvae . In contoisduval  and morem Herbst . Regardim Herbst ,86,87. SAmmi visnaga L. may inhibit ecdysone and further affect the activity of acid phosphatases and molting of Schistocerca gregaria Forsskål nymphs [Comparisons of biological activity of anise, dill and fennel EOs with commercial insecticide neem revealed higher toxicity and antifeeding activity of EOs, and stronger molting delay effects of neem. It has been proved that the neem dominant component azadirachtin is very effective to inhibit the synthesis of active molting hormone 20-hydroxyecdysone, disrupts growth, may lead to incomplete ecdysis, malformations in pupae and adults and reduced fecundity . EOs mayl nymphs .T. castaneum [Spodoptera frugiperda J.E. Smith they might be involved in delayed pupation through inhibition of tyrosinase and cuticle synthesis [We showed that EOs of anise and fennel were rich in phenylpropanoid trans-anethol and dill EO was rich in oxygenated monoterpene carvone and monoterpene hydrocarbons α-phellandrene and limonene. High toxicity and antifeeding activity in insect pests are induced by major compounds of anise, dill and fennel EOs: trans-anethol ,94,95 anastaneum . In Spodynthesis ,101. Howynthesis . Besidesynthesis ,104,105.Physiological mechanisms of insecticidal activity of EOs, their compounds and/or a mixture of compounds is to act as neurotoxins and lead to the paralysis and death of insect pests through the inhibition of acetylcholine esterase (AChE), as well as through blockage of octopamine receptors and/or interference with GABA-gated sodium channels . For exaT. castaneum adults exposed to anise EO [P. unipuncta larvae exposed to trans-anethol [Obviously, exposure to EOs and EOs compounds induce numerous changes in physiological processes that may further affect pest insect behavior and life-history traits. In the present paper, we assessed how three Apiaceae EOs affected the growth and nutritional indices of GML. We showed mainly significant adverse effects of EOs and neem on GML growth (RGR), consumption (RCR), assimilation (AD) and metabolism . In difference to neem experimental group, larvae fed on 0.5% EO supplemented diet lost their mass and had negative values of growth and gross/net growth efficiencies . Therefore, GML mass change during 2 days of feeding was provoked not only by among-treatment variation in the amount of consumed and assimilated food  but also was a consequence of the EO influence on post-ingestive and post-digestive mechanisms. Our results are in accordance with findings of other studies which showed negative values of RGR and ECI in anise EO  and 4th -anethol . Reduced consumption obtained in the present paper in 2nd and 4th instars fed on anise, dill and fennel EO treated diets as well as in studies on the effects of other EOs on GML ,70,74,75Inula racemosa Hook which contained sesquiterpene lactones did not affect RCR and RMR of Spodoptera litura Fabricius larvae at the lowest examined concentration, whereas both indices were reduced at the highest concentration [P.unipuncta exposed to trans-anethol and EO from Petroselinum crispum (Mill.) Nyman ex A.W. Hill, two botanicals which induced larval mass loss. Despite the decrease in the amount of metabolized food in GML fed on EO- and neem-supplemented diets, its proportion relative to ingested and assimilated food increased leading to lower ECI and ECD values.Our results showed that RMR decreased along with a decrease in RCR. Such response depended on concentration so that RMR and RCR were not significantly changed at the lowest concentration of anise and fennel EOs. Likewise, extracts of ntration . Sousa entration  recordedS. frugiperda and Anticarsia gemmatalis Hübner limonene, carvon, estragol and anethol  are detoxified by microsomal cytochrome P-450 monooxygenases [Reduction in food utilization efficiency by botanical treatments points to their chronic toxicity which forces larvae to reallocate energy resources from growth to defense. Depletion of metabolites and adaptive increase in antioxidative and detoxification enzymes have been described in pest insects exposed to EOs ,125,126 ygenases . This enygenases .Pimpinella anisum L.), dill (Anethum graveolens L.), and fennel (Foeniculum vulgare Mill.) cultivated on the experimental fields of the Institute for Medicinal Plant Research “Dr. Josif Pančić” in Pančevo, Serbia . EOs were obtained by hydrodistillation of their seeds using a Clevenger-type apparatus [Tested EOs have been extracted from seeds of anise (pparatus .−1) and 1 μL of each solution was injected in a split-mode (1:30). Gas chromatography was performed using the GC Agilent Technologies 7890A apparatus equipped with a split-splitless injector attached to an HP-5 column  and fitted to a flameionization detector (FID). The operating conditions were: the carrier gas was H2 (1 mL/min/210 °C); the temperatures were set as follows: injector at 250 °C and detector at 280 °C, while the column temperature was linearly programmed from 40 to 260 °C at 4 °C/min. The percentage composition was computed from the peak areas, without correction factors. The essential oils samples were diluted in ethanol  and by comparing their experimentally determined retention indices  to the data from the literature [The Gas Chromatography—Mass Spectrometry was performed using the HPG 1800 C Series II GCD analytical system equipped with an HP-5MS column . The carrier gas was He (1 mL/min). Other chromatographic conditions were the same as those for Gas Chromatography with Flame-Ionization Detection. The transfer line was heated at 260 °C. The mass spectra were recorded in the EI mode (70 eV) in the range of terature .Gypsy moth egg masses were collected from natural populations in the Lipovica Forest, near Belgrade, Serbia  during the autumn. Egg masses were maintained at 4 °C until the next spring. Eggs were mechanically cleaned of hairs, disinfected by soaking into 0.1% sodium hypochlorite solution for 5 min, washed with distilled water for 10 min and air-dried . Eggs frQuercus robur L.) leaf disc (30 mm diameter) in the center of the Petri dish in the no-choice test or two discs on opposite sides of the Petri dish in the choice test. Leaf disc treatments were performed by the leaf dipping method [®-T/S (Trifolio-M GmbH) at three different concentrations  or in a solvent for 3 s. In the no-choice test leaf discs were treated with the EOs, neem or solvent, whereas in the choice test, one leaf disc was treated with the EOs or neem and the other with the solvent. After 30 minutes’ evaporation of the solvent, leaf discs were fixed to the agar layer with pins. Then one larva was introduced into the center of each Petri dish. After 48 h, the remains of the consumed discs were scanned at 200 dpi in jpg format. Quantification of the consumed surface area for each leaf disc was done by subtracting remained leaf disc area from the disc area at the start of the experiment using ImageTool software 3.0 [The antifeeding activity was assessed in the 2nd instar larvae by no-choice and choice tests. After the molting into the 2nd instar, larvae were starved for 24 h. An agar-water (2%) layer of 2 mm thickness was poured into Petri dishes (90 × 14 mm). After agar turned solid we covered it with wet filter paper and placed one oak , relative (RDC), and total (Tot) deterrence coefficients were calculated according to the formulas of ,139:ADC The total deterrence coefficient can range from −200 to +200. According to Tot values EOs can be ranged as very good , good (101–150), average (51–100), and weak deterrents (˂50). Negative Tot values suggest attractant properties of EOs. In the digestive toxicity test, EOs and neem were incorporated into the artificial diet at different concentrations . After starvation for 24 h, ten 2nd instar larvae were put into the Petri dishes and fed ad libitum either on control diet or on EOs and neem treated diets for 24 h. After that, they were transferred into clean Petri dishes and fed on control diets for another 96 h (120 h from the beginning of exposure). During the experiment, two fresh cubes of artificial diet per Petri dish were provided daily. Within each EO or neem and their concentration five replicates were analyzed . Larval mortality and molting were monitored daily and 120 h after the beginning of experiment, the percentage of mortality and percentage of molting into the 3rd larval instars were determined. The percentage of molting reduction relative to the control was calculated as (C − T)/C × 100 where C was the mean percentage of molted larvae in the control group and T was the percentage of molted larvae fed on EO or neem treated diet.After molting into the 4th larval instar, larvae were separated and exposed to 24 h starvation after which their mass was measured individually. Larvae were daily supplied with cubes of the artificial diet with incorporated EOs or neem. As preliminary study showed that larvae fed on EO supplemented diets decreased their mass at the concentration of 0.5%, the study of EO and neem influence on nutritional physiology of GML encompassed concentrations of 0 (control), 0.1, 0.25, and 0.5%. Cubes of artificial gypsy moth diet were weighed before and after the feeding trial, as well as the excrement was weighed at the end of the experiment. Larval mass was measured again 48 h after the experiment was set. All indices were estimated on a dry mass basis. Larvae, uneaten cubes and excrements were dried at 65 °C for 72 h. After this time, the mass of each larva, uneaten cubes, and excrements were weighed. A regression of dry on fresh mass in a random sample of 30 larvae and cubes of artificial diet per experimental group was used for estimating the dry mass of larvae and cubes of artificial diet at the beginning of the experiment. Based on these data the following indices were calculated according to the standard formulas ,141,142 0.5 transformed values of the absolute antifeeding index, larval mortality, larval molting reduction, and log-transformed values of the amount of consumed food, amount of assimilated food, and amount of metabolized food. Dunnett test following one-way ANOVA was used to estimate the significance of differences of treatment groups from the control group. Two-way ANOVA was carried out to evaluate the main and interaction effects of the botanical type (EOs or neem) and the botanical concentration as fixed factors on examined traits. A least square means (LSM) test with the Bonferroni correction was used for a posteriori comparisons (contrasts) of different concentration effects within each EO and neem as well as comparisons of EOs and neem effects within each concentration. Parametric one-way and two-way ANOVAs were performed by software package Statistica 7.0  on untransformed values of the relative deterrence index and (X + 0.5)p ˂ 0.0001). Therefore, significant differences of treatment groups from the control group were revealed by Welch one-way ANOVAs for each pair of comparison followed by the Bonferroni correction [For mass gain, and growth and nutritional indices assumption of homogeneity of variances was strongly violated (Levene’s test, rrection . Signifirrection . LS meanIn whole, Apiaceae EOs can be considered as promising strategy for gypsy moth control based on strong negative effects on survival and consumption in 2nd instar, and impairment of nutritional physiology in 4th instar larvae. Whereas anise EO was the best antifeedant, dill EO exhibited the highest mortality. At a concentration of 0.5%, the three Apiaceae EOs were more effective than commercial insecticide neem in reducing RGR, ECI, AD and ECD of 4th instar GML. Anise, dill and fennel are spice plants used in human nutrition, and have low toxicity to mammals and other non-target organisms ,147,148.


To overcome graphene loss, we investigated 2-D hexagonal boron nitride (h-BN) as an alternative. Unlike graphene on AlN, it was confirmed that h-BN on AlN was intact after the same high-temperature process. Moreover, the overgrown AlN layers on both h-BN/AlN and h-BN/Al2O3 could be successfully exfoliated, which indicates that 2-D h-BN survived after AlN growth and underlines its availability for the vdWE/RE of III-nitrides with further mechanical transfer. By enhancing the stability of the 2-D material on the substrate, our study provides insights into the realization of a novel epitaxy concept.A challenging approach, but one providing a key solution to material growth, remote epitaxy (RE)—a novel concept related to van der Waals epitaxy (vdWE)—requires the stability of a two-dimensional (2-D) material. However, when graphene, a representative 2-D material, is present on substrates that have a nitrogen atom, graphene loss occurs. Although this phenomenon has remained a hurdle for over a decade, restricting the advantages of applying graphene in the growth of III-nitride materials, few previous studies have been conducted. Here, we report the stability of graphene on substrates containing oxygen or nitrogen atoms. Graphene has been observed on highly decomposed Al A challenging approach, but one providing a key solution to material growth, remote epitaxy (RE)—a novel concept related to van der Waals epitaxy (vdWE)—requires the stability of a two-dimensional (2-D) material. Recently, the results of applying graphene at an interface similar to that of the homo-epitaxy have been reported using gallium arsenide (GaAs)/graphene/GaAs,21 zinc oxide (ZnO)/graphene/ZnO,22 and strontium titanate (SrTiO3)/graphene/SrTiO3;23 the structures of these materials are a result of the interaction between the grown material and the substrate. This structure  is called remote-epitaxy (RE); specifically, the exfoliation of the overgrown material occurs as a result of the weak interaction at the interface, and the dislocation-free structure is caused by graphene transparency.24 In conventional GaN growth on SiC, Si (111), and Al2O3, the high dislocation density results in a critical problem owing to lattice mismatch, which typically deteriorates the device quality. Additionally, the strong chemical bond between the epitaxially grown GaN and conventional substrates limits the possibility of using this material in various fields of application. RE can pave the way to overcoming the problems of conventional GaN growth; however, the application of RE has been very challenging for materials containing a nitrogen (N) atom, such as the structure of III-nitride/graphene/III-nitride. It has been almost a decade since the research on GaN growth on graphene was reported.6 Meanwhile, in our previous study, it was highlighted that if a substrate supporting graphene has a N atom , graphene-loss occurs under a conventional growth environment, unlike other reported N-free substrates.25,26 This graphene-loss problem remains unsolved and must be overcome in order to realize RE on III-nitride materials.Placing a two-dimensional (2-D) material at the interface between a grown material and a substrate opens novel opportunities for semiconductor devices. The epitaxy caused by a 2-D material, termed van der Waals epitaxy (vdWE), reduces the dislocation density of the grown material, which strongly affects the device properties, and also allows exfoliation of the grown material from the substrate owing to the weak chemical bond.3 bonds on graphene. Recently, N-doped graphene with carbon (C)–N bonding has been demonstrated by annealing in ambient ammonia (NH3)7,27 and using N2 plasma treatment.13,16 Feng et al. have successfully created a GaN/graphene/SiO2 structure using ambient NH3 annealing on graphene.7 Chen et al. have also shown UV-LED growth on graphene/Al2O3 using N2 plasma treatment before growth.16 These results confirm that N-doped graphene on a substrate containing an oxygen (O) atom is a promising approach to enabling vdWE. Moreover, Jeong et al. have recently shown that graphene is stable on a substate containing an O atom  and that the substrate can be reused for the remote heteroepitaxy of a GaN microrod light emitting diode (LED).18 Meanwhile, regarding the RE structure of III-nitride/graphene/III-nitride, the stability of the 2-D material on the decomposed III-nitride substrate warrants investigation because the growth temperature of III-nitride is higher than its decomposition temperature,28 as shown in Fig. S1a,As graphene has an extremely low surface energy, it is difficult to directly grow III-nitride materials upon it. To allow III-nitride growth on graphene, a potential approach is to generate sp2O3 and AlN were used as substrates to support the graphene. Graphene could still be observed on even the highly decomposed Al2O3 at over 1200 °C, but disappeared on the decomposed AlN at over 1300 °C. Conversely, h-BN grown on AlN was intact after annealing at 1400 °C. We further demonstrated that the overgrown AlN layers on both h-BN/AlN and h-BN/Al2O3 could be successfully exfoliated, which indicates h-BN is robust during metal–organic chemical vapor deposition (MOCVD) of III-nitrides and could be used as an alternative for vdWE.Here, we report the results of an investigation into the cause of graphene-loss and suggest an alternative 2-D material for vdWE or RE of N-containing III-nitrides. We investigated the stability of two different 2-D materials, graphene and h-BN, on substrates containing either N or O as shown in Fig. S1b2O3 and AlN and annealed over 1100 °C for 10 min in ambient hydrogen (H2) by MOCVD. Previous studies on the influence of H2 on graphene have shown that H2 passivates broken points of graphene accompanied by dangling bonds, which could help move a large atom, such as gallium,29 aluminum,30 and indium31 under graphene; however, the aforementioned studies also show that H2 does not result in graphene-loss. Therefore, we used H2 to obtain substrate decomposition at a relatively low temperature rather than ambient inert gas. The photograph of the annealing results shown in 2O3 annealed at each temperature is observed as a black sheet; however, there is no trace of graphene on AlN annealed at 1400 °C. Therefore, we investigated why and how the graphene-loss occurs on AlN. 2O3 and AlN was evaluated using Raman measurements. To analyse the effect of substrate decomposition in detail depending on the temperature, we reveal the fitting data of the D, D′, and 2D peaks at 1300–1700 cm−1 in Fig. S2.2O3 close to 1350, 1580 and 2700 cm−1, respectively. With an increase in the annealing temperature, ID/IG also increases. These defect points are closely related to the decomposition of Al2O3, the critical temperature point of which is approximately 1200 °C in ambient H2.32 Over 1200 °C, the aluminium (Al)–O bond is broken and Al, and O atoms are generated. This O atoms create the D and D′ peaks of graphene, which are similar to those of the O2 plasma-treated graphene.33,34 It is also accurately demonstrated that Al generated by Al2O3 decomposition does not lead to graphene-loss because graphene is observed across the whole surface of all samples over 1200 °C. From the Raman data alone, as shown in 2O3 substrate affects the D and D′ peak of graphene, but it does not lead to graphene-loss. However, in the case of the decomposed AlN containing the same atom, Al, even though it has a much higher decomposition temperature than Al2O3,35 graphene completely disappears at 1400 °C. Regarding the result of annealing at 1300 °C, graphene only partially remained on AlN, as shown in the scanning electron microscopy (SEM) images shown in Fig. S5.−1, which was attributed to the AlN peaks by the reference data shown in Fig. S6.2O3 surface gradually became rough as the annealing temperature increased, graphene wrinkles were still observed, even at 1400 °C. In contrast, on AlN at 1300 °C, the starting temperature of AlN decomposition, graphene wrinkles and the decomposed surface were simultaneously observed, which means that graphene could disappear owing to the decomposition of AlN containing a N atom. The voids were commonly observed on Al2O3 and AlN from 1200 and 1300 °C, respectively, and the void size increased along with the annealing temperature, which implies that voids were generated by substrate decomposition.32,35,36 Moreover, as shown in Fig. S7,2O3 at 1400 °C in ambient inert gas  as shown in Fig. S8.2, the decomposition of AlN was negligible in ambient inert gas, even though the temperature reached 1400 °C.35 As a result, graphene was observed on the AlN layer after the annealing process. Moreover, using Raman spectroscopy we also confirmed that graphene on Al2O3 is partially oxidized into graphene oxide after the same annealing process. This observation is consistent with a previous report by Akiyama et al., in which the authors found that marked decomposition of Al2O3 starts at around 1450 °C.32 It is therefore reasonable to suggest that the graphene oxide observed in this study resulted from the O atom arising from the slightly decomposed Al2O3 owing to the reduced 2D peak intensity, unlike graphene on AlN.To investigate the stability of graphene on a substrate at high temperatures, graphene was transferred to Alet al. have recently reported that N-plasma could result in N incorporation in the graphene lattice. Moreover, they demonstrated decreasing X-ray photoelectron spectroscopy (XPS) intensities of C 1s after graphene was exposed to N-plasma.92O3, demonstrating that the atomic percentage of C is relatively constant, which clearly differs from that in ID/IG and  and is thus very likely to be doped with N. Many reports showed the binding energy of the C–N bonds by measuring XPS. The N-sp2 C, N-sp3 C, and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O peak positions are mainly reported at 284.9–285.9, 286.5–287.5, and 288.9–289 eV respectively.16,37–45 As shown in 2 C, N-sp2 C, N-sp3 C, and CO respectively. Meanwhile, Ryu et al. demonstrated that the decomposition of GaN generates C–N bonds in graphene oxide, but also incorporates N atoms into graphene oxide,46 which could occur during the annealing of graphene on AlN over 1300 °C in this work. In general, the N 1s of N-doped graphene is characterized by three peak positions, these are, pyridinic-N (398–399 eV), pyrrolic-N (400–401 eV), and graphitic-N (401–402 eV).7,16,37–45,47 Recently, Chen et al. demonstrated a AlN/graphene/sapphire structure using N-doped graphene.48 They observed N-doped graphene with AlN and showed sp2 C–N, and Al–N bonds in the N 1s scan using XPS, although N-doped graphene was covered by AlN. Similar findings were also observed in the N 1s spectrum, as shown in 49 Moreover, the peak expected for pyridinic-N was observed at 398.2 eV, which implies that the N atom could be incorporated in the graphene lattice. A schematic diagram of the graphene structure affected by the N atom is shown in 37,45,47,50 Additionally, with an increase in the N-doping concentration, the Raman peaks of graphene gradually fade.37,45,47 Compared with the reported N-doped graphene model based on our results, the N atom generated by the decomposed III-nitride substrates could also produce the N-doped graphene, which creates many vacancy areas with a loss of C and, thus the elimination of graphene. The O atom creates defect points on the graphene as sp3 bonding and changes graphene to graphene oxide;51 however, the influence of the O atom on the graphene-loss is not critical as comparing to that of the N atom, as shown in †When the annealing temperature reached 1400 °C, the surface was fully decomposed, and graphene completely disappeared on AlN. It is important to understand why and how graphene-loss occurs on the substrate containing an N atom. We speculate that graphene-loss is closely related to the highly N-doped graphene structure. Barbier 19,20 For comparison, we also examined the stability of the h-BN film on AlN using an identical annealing process to that used for graphene on AlN in this work. The AFM images in 2 for 10 min. Clearly, there is almost no difference between these two h-BN surfaces, and the root mean square roughness of the surface before and after annealing is 1.167 and 1.114 nm, respectively. Both the surfaces are decorated by surface wrinkles with a few tiny holes arising from insufficiently coalesced h-BN domains. The h-BN samples were further examined using Fourier transform infrared (FTIR) spectroscopy in the reflection mode. Meanwhile, the AlN/sapphire template was also characterized prior to h-BN deposition. As shown in −1, which is correlated to the transverse phonon frequency of AlN.52 Moreover, both the as-grown h-BN and annealed one exhibited a clear vibration mode at approximately 1368 cm−1, which is associated with the in-plane transverse phonon mode of the sp2-bonded h-BN.53As reported in previous studies, the 2-D h-BN layer serving as a platform for III-nitrides vdWE and/or RE could be another option to overcome the stability issue of graphene on a substrate that contains the N atom discussed above.2O3 peak because it was measured on the Al2O3 substrate after the exfoliation process. Furthermore, Specifically, the peak features for these two h-BN samples are almost the same, which indicates that the effect of the high-temperature (1400 °C) annealing process on the as-grown h-BN film on AlN is negligible. Based on these results, it was confirmed that 2-D h-BN is more stable than graphene on a substrate containing nitrogen. To further examine the stability of h-BN for subsequent AlN overgrowth, around 800 nm-thick of AlN was grown on a template of 5 nm-thick h-BN film on AlN/sapphire. c-plane Al2O3, in which the formed AlN layer almost coalesced on h-BN/c-plane Al2O3, and this can even be easily exfoliated using scotch tape, as shown in c-plane Al2O3 presents an island-like surface of the AlN layer, as shown in Fig. S11,In addition to the AlN overgrown on h-BN on AlN, we also examined the AlN growth on 3 nm-thick h-BN on sapphire and demonstrated the successful post-growth exfoliation of AlN. 2O3, graphene was observed even though Al2O3 was fully decomposed; however, graphene was not observed on the decomposed AlN, which means that the N atom generated by substrate decomposition not only causes graphene loss, but also limits the application of graphene in novel epitaxy concepts. Unlike graphene on AlN, layered h-BN grown on AlN exhibited good stability and did not show significant differences in surface features before and after the annealing process. Furthermore, it was also observed that the grown AlN layers on h-BN/AlN and h-BN/Al2O3 could be easily exfoliated. All of these results prove that h-BN is stable on the AlN surface and survives during the high-temperature AlN overgrowth process. Therefore, it is necessary to use a substrate that can avoid graphene loss and to select appropriate 2-D materials, such as h-BN, that are sufficiently robust for the realization of a novel epitaxy concept for III-nitrides.In conclusion, we investigated the stability of graphene on substrates containing O or N atoms and demonstrated that graphene was unstable on substrates containing an N atom above its decomposition temperature. In the case of graphene on AlH. Amano and D.-S. Lee directed and funded this work. J.-H. Park and X. Yang conceived and wrote the manuscript. J.-H. Park, X. Yang, J.-Y. Lee, M.-D. Park, and M. Pristovsek conducted experiments. J.-H. Park, X. Yang, J.-Y. Lee, M.-D. Park, S.-Y. Bae, and M. Pristovsek analysed data. All authors discussed the results and commented on the manuscript.There are no conflicts to declare.SC-012-D1SC01642C-s001


Reduced graphene oxide (rGO) flakes obtained by thermal annealing of graphene oxide at 300 and 600 °C exhibited electrical conductivities of 2.3 ± 1.0 and 14.6 ± 5.5 S/m, respectively, showing the effect of thermal reduction on the electrical conductivity of rGO flakes. This study demonstrates an alternative method to characterizing the intrinsic electrical conductivity of graphene-based materials, which affords a clear understanding of the local properties of individual graphene flakes.The intrinsic electrical conductivity of graphene is one of the key factors affecting the electrical conductance of its assemblies, such as papers, films, powders, and composites. Here, the local electrical conductivity of the individual graphene flakes was investigated using conductive atomic force microscopy (C-AFM). An isolated graphene flake connected to a pre-fabricated electrode was scanned using an electrically biased tip, which generated a current map over the flake area. The current change as a function of the distance between the tip and the electrode was analyzed analytically to estimate the contact resistance as well as the local conductivity of the flake. This method was applied to characterize graphene materials obtained using two representative large-scale synthesis methods. Monolayer graphene flakes synthesized by chemical vapor deposition on copper exhibited an electrical conductivity of 1.46 ± 0.82 × 10 Since the intriguing electronic properties of graphene have been experimentally observed in mechanically cleaved graphene ,2,3, it Electrical conductivity is one of the most important properties of synthesized graphene. However, the intrinsic electrical conductivity of graphene is significantly affected by its atomic and chemical structures. For example, the presence of grain boundaries in polycrystalline CVD-grown graphene deteriorates its electrical conductivity owing to the scattering at the grain boundary interfaces . GraphenThe electrical properties of monolayer graphene have been investigated using several methods. To characterize individual graphene flakes, micro-sized metal electrodes were patterned on a graphene flake using photolithography techniques. Although this method has been extensively used to study the electrical properties of graphene flakes, such as mechanically exfoliated graphene and individual GO flakes ,12, it rIn this study, we employed C-AFM to directly measure the electrical conductivity of individual graphene flakes prepared by two prominent scalable synthesis methods: CVD on copper and the thermal reduction of GO. Without using any additional materials for electrical measurements, a graphene flake connected to a pre-fabricated electrode was scanned using an AFM tip, which generated an in-plane current distribution. The current change was analyzed as a function of the distance from the electrode to extract the electrical conductivity of the individual graphene flakes. This method was applied to CVD-grown monolayer graphene flakes. Furthermore, individual rGO flakes were characterized as a function of the degree of thermal reduction.High-quality sub-monolayer graphene was synthesized on copper foil using low-pressure chemical vapor deposition (LPCVD) . Copper 2/Si . T. T2-hybr20 cm−1) ,46,47,48l groups ,50, whic2 (Wt = 4.872 nm) and 20.750 nm2 (Wt = 5.140 nm) at annealing temperatures of 300 and 600 °C, respectively. Therefore, using the given equation to fit the resistance–distance curves, the electrical conductivities were estimated to be 2.3 ± 1.0 and 14.6 ± 5.5 S/m for 300 and 600 °C annealing samples, respectively (D/IG. Several previous studies have investigated the electrical conductivity of isolated graphene flakes. Monolayer rGO flakes with a C/O ratio of ~4 synthesized by the photoreduction of GO using ultraviolet light irradiation in vacuum exhibited an electrical conductivity of 0.20 S/m, which was directly measured by C-AFM [ectively f. This iby C-AFM . Moreoveby C-AFM . A similby C-AFM . Conside2/Si exhibited a high electrical conductivity comparable to that measured by the van der Pauw method. In addition, GO flakes placed on SiO2/Si were thermally reduced at two different temperatures. Annealing the rGO flakes at higher temperatures resulted in a higher electrical conductivity, which was confirmed by an increase in the C/O ratio and a decrease in ID/IG. This study demonstrates that the C-AFM method is capable of estimating the local electrical conductivity of individual graphene flakes. In addition, this study provides a better understanding of graphene materials synthesized using the two major scalable production methods.The electrical conductivities of isolated graphene flakes were investigated using C-AFM and an analytical analysis. Monolayer graphene flakes obtained by CVD on copper foil and the thermal reduction of GO were characterized using C-AFM. High-quality CVD-grown monolayer graphene flakes transferred onto SiO


Ammonia emissions were product of ammonia concentration and air exchange rate. Temperature and relative humidity were measured to establish microclimate conditions in buildings tested to show the overall microclimatic situation in buildings. Differences between ammonia emission rates were observed in both housing systems. The highest ammonia emission rate was equal to 2.75 g·h−1·LU−1 in well-ventilated cattle barn with the largest herd size.The aim of the study was to present the scale of greenhouse gas emissions from animal production, and to provide test results from different housing systems. In three free stall buildings, two with slurry in deep channels and one with cattle in cubicles staying on shallow litter concentration of ammonia and carbon dioxide were measured in summer season by using dedicated equipment from Industrial Scientific Research. Air exchange was calculated on the base of balance carbon dioxide method. This method was used in order to estimate the air flow rate. Concentrations of ammonia and CO Milk and meat production are finally balanced with an environmental and animal welfare conditions to minimize negative influence for the environment. Major amount of nitrogen are leaching from livestock production to the environment.According to the inventories, agriculture is a significant source of greenhouse gases (GHG) . Nosek et al.  confirme2, SF6 or cryptone 85 as tracer gases  by using gas concentration meters, made by company Industrial Scientific Co.V) using validated method of carbon dioxide balance. For metabolic emission of carbon dioxide by one LU, average values were used WCO2 = 220 g·h−1·LU−1 according to the Institute of Zootechnics in Cracow.Estimation of air exchange rate (E).Calculation of ammonia emission (The methods in our research consisted of the following stages:E) was equal product of air exchange rate (V) and ammonia concentration (S):Ammonia emission , connected with concentrator LB-731 for data collecting.The following measurement equipment was used:A short characteristic of herd like herd size and system of removing manure is shown in Table The obtained results of harmful gas emission which is ammonia depend on the effectiveness of the ventilation.−1·LU−1). In contrary, the lowest emission 1.47 (g·h−1·LU−1) was observed in a cattle barn with the lowest cubage.The highest level of ammonia emission was observed from cattle barn with deep slurry channels and with the highest ventilation rate which amounted 2.75  was below than critical level of 0.05 (5%), and the statistical empirical value F = 27.494. The statistical analysis of the temperature influencing the NH3 emission level delivers that the significance level (p) was 0.01184 and the statistical empirical value F = 4.4595. The case of temperature impact on the CO2 emission level, inversely than the NH3 emission level, delivers the correlation. The obtained results were the basis for the Duncan tests that determine the temperature values to homogeneous groups. The analysis showed that each of the tested temperatures is in a different homogeneous group, which makes significant differences in the temperature impact on the level of CO2 emissions. The mean temperature for the low level of the CO2 emission was 21.5 °C, for the average level was close to the 23.2 °C, but high emission was not known. The correlation of relative humidity outside and relative humidity inside with the NH3 emissions from cattle houses was conducted. The estimated recommended levels of NH3 emissions were also used. The results of relative humidity outside and humidity inside correlation with the CO2 and NH3 levels were presented in Fig. During the statistical analysis, the expected marginal mean of temperature influence to CO2 and NH3 emission levels in both cases. Similarly, in both statistical analyses, the significance value (p) was below a critical level, which means that the correlation exists. The empirical value of statistics F during the relative humidity outside and relative humidity inside comparison with the CO2 was equal to 34.726, and the Wilks Lambs = 0.92405. In the case where the relative humidity outside and relative humidity inside were correlated with NH3, the empirical value of statistics F = 17.507 and the Wilks Lambs = 0.92201. The characteristics of homogeneous groups defining the effect of relative humidity outside and relative humidity inside comparison with the CO2 and NH3 levels were presented in Table It was statistically confirmed that the relative humidity outside and relative humidity inside had an influence on the COcs F2, 84 during t2 and NH3 emission. Statistical analysis confirmed the need for reducing the relative humidity inside to limit the CO2 and NH3 emissions. Considering the whole scope of the conducted studies, it can be noticed that the best conditions for limiting CO2 are to reduce temperature and humidity outside and inside of livestock housing. Ammonia emission could be reduced by simultaneously decreasing air humidity and decreasing air temperature. In the case of NH3 emission, reduction is necessary to increase the temperature and reduce the humidity inside the building.According to Table 3 emissions were observed. A similar situation was described by Zhang who tested ammonia emissions from 11 types of cattle barns, with different floor and manure removing systems and the highest emission was in non-littered cattle barns  despite weather conditions. In particular, in non-litter cowsheds, higher NHSimilar results were obtained by a Polish researcher, which calculated ammonia emissions by using model (not measured) from dairy cattle for particular technologies ranging from 6.4 per year for deep litter up to 28.69 kg per year for a slurry system, but these results based only on simply assuming fixed rate of nitrogen losses from manure in livestock buildings   compared to other authors’ results.Table 3 emission from litter-free barns was about two times higher than emission from barns with litter. A similar trend was obtained in our research, where the emission from the litter-free system in one of the barns was about 24 kg·year−1·LU−1∙and for the litter system 12.87 kg·year−1·LU−1.According to Demmers et al. , the annRecently, livestock production significantly increased in Central Europe that involved the need of correction of emission factors. Generally, in Poland, it is utilized emission coefficient elaborated in Northern European countries . In this study, first step was made to present Polish emission factor dedicated especially to summer season conditions.2 and NH3 emission. A completely different validity occurred in accordance to the measured temperature values, where the increase in temperature could cause the reduction of NH3 emission. However, this validity was not confirmed by statistical analysis where the lack of temperature influence on the NH3 emission level confirms the value significance level p = 0.01184. All other cases of statistical analysis have reached the significance value p below the critical level of 0.05. The main conclusion from the research is that ammonia emissions from cattle barns with slurry were higher than from cattle barn bedded with straw, but simultaneously in the night period, both emission levels were comparable.Although the change of temperature and humidity was not huge, measured values allowed estimating the levels of gas emission in order to carry out the statistical analysis. According to the study, the increase of relative humidity outside and relative humidity inside caused an increase of CO


Two-dimensional (2D) materials and their composites have gained significant importance as the functional layer of various environmental sensors and nanoelectronics owing to their unique properties. This work reports for the first time a highly sensitive, fast, and stable humidity sensor based on the bi-layered active sensing area composed of graphene flower (GF) and poly  PVA thin films for multifunctional applications. The GF/PVA humidity sensor exhibited stable impedance response over 15 days, for a relative humidity (RH) range of (40–90% RH) under ambient operating conditions. The proposed bi-layered humidity sensor also exhibited an ultra-high capacitive sensitivity response of the 29 nF/%RH at 10 kHz and fast transient response of 2 s and 3.5 s, respectively. Furthermore, the reported sensor also showed a good response towards multi-functional applications such as non-contact skin humidity and mouth breathing detection. Thin film-based capacitive and impedimetric sensors offer numerous benefits over optical and chemical sensors in vapor detection applications, namely low energy consumption, higher sensitivity response and selectivity, good stability, and fast response/recovery time ,2. In inCarbon-based materials, such as graphene and carbon nanotubes (CNTs), have sparked a great deal of interest in chemical sensing because of their distinctive structural and electronic properties, such as a large surface area, compact size, high electronic mobility, and high sensitivity response to electrical interruptions from water or other gaseous molecules ,20,21,22Polymers are another class of material with a high sensitivity to many environmental factors. Low cost, simple synthesis, high stability, and good mechanical characteristics for flexible electronics are just a few of the major advantages of utilizing these materials ,32,33,342/g, transparent nature, remarkable conductivity, and highly crystalline structure [Herein, for the first time, we demonstrate a unique sorption layer (multi-layered graphene nanostructures known as graphene flower deposited over PVA film), used to fabricate a fast, highly responsive, and extremely sensitive humidity sensor for future applications in printed electronics. GF material has attractive properties such as a large surface area of 500–2500 mtructure ,45,46,473, and metal content ~70 ± 2 wt% from InkTec, Ansan-si, Gyeonggi-do, Korea. We purchased the 100-µm-thick PET substrate from AgIC paper. The PVA solution was formed by mixing PVA powder in the DI Water to 1:10 mass ratio and kept at room temperature for 24 h to swell. The solution was then magnetically stirred by keeping it on the hot plate for 12 h at 343 K to dissolve PVA completely as seen in GF solution in propylene glycol monomethyl ether acetate (PGMEA) (99.9 wt%) was purchased from InALA . PVA polymer was purchased from Sigma-Aldric . For the electrode’s preparation, we purchased screen-printing Ag ink with viscosity 155,000 ± 15,000 cps, density ~2.8 ± 0.2 g/cmThe scanning electron microscope TESCAN MIRA 3  was used to analyze the surface morphology of the deposited active material, while dispersive X-ray (EDS) spectroscopy was used to investigate the elemental composition. The crystal structure of GF and its composite with PVA was studied using an X-ray diffractometer . Fourier transform infrared (FTIR) spectroscopy was performed by using Fourier Transform infrared spectrometer ALPHA II . Additionally, Raman spectroscopy was performed using Micro Raman Spectrometer System LABRAM HR EV .rms source alternating voltage signal. When the RH of the chamber changed the capacitance and impedance spectra incorporating the impedance and capacitance responses were acquired by interfacing the LCR meter with a personal computer. The proposed sensor’s humidity responses were recorded over a wide humidity range, and the temperature was kept constant at 22 °C during the experiment. The test frequency was maintained constant at 1 kHz and 10 kHz while examining the sensor’s electrical response. The humidification level of the chamber was gradually increased while recording the measurement to get the maximum number of data points.We studied the electrical response of the sensor by exposing it to varying levels of RH over a wide range. θ = 27°), in θ = 25.8° disappears in the composites as the graphene flower is dispersed into the PVA matrix, implying graphene disorder and loss of structural regularity and intercalation of PVA into inter-layers of graphene. As a result, the graphene nanostructures are thought to have been disseminated into the PVA matrix on a molecular level. In the composites, similar results were reported for studies performed previously, by adding graphene nanostructures in the PVA with different volume ratios. To determine the purity of the materials and study their probable interaction, Fourier transform infrared spectroscopy (FTIR) was used to investigate the functional groups in active layer materials, as shown in −1 originating from C–H stretching vibrations. From the FTIR spectrum of PVA, peaks at 1450 cm−1 (C–H bending) and1100 cm−1 (C–O–C stretching) can be observed, confirming the presence of pure PVA in the composite. Small peaks positioned at 1376 cm−1 can be attributed to C–H bonds, while at 1260 is specific to C–O bonds, and a small peak at 839 cm−1 is attributed to C–C stretching vibration. Chemical composition of the sensor film was determined using Raman spectroscopy (LabRAM HR Raman). −1, 1575 cm−1, and 2696 cm−1, respectively. The 3D Nano Profiling System (NV2200) was used to find out the surface roughness of sensing active material. From this 3D view, the thickness of PVA/GF, and GF was estimated to be 4.7 µm, and 2.1 µm respectively as shown in To examine the surface morphology of the proposed humidity sensor’s PVA/GF composite thin film, a scanning electron microscope image was taken of the active layer, as shown in We measured the impedance and capacitance responses under different humidity levels at test frequencies of 1 kHz, and 10 kHz to explore electrical performance of the proposed humidity sensor. RHC and RHoC are the capacitances at %RH and 40% RH, respectively. + ions will rise under these conditions, and they will be able to hop between water vapor molecules (Grotthuss mechanism). When the amount of water vapor in the atmosphere increases, more H+ ions migrate, causing the PVA/GF conductivity to rise. Protons are the primary carriers responsible for electrical conductivity when exposed to moisture.The capacity of PVA to sense moisture is due to an OH group, which can be seen in the PVA structure. The capacity of GF to identify water molecules is dependent mainly upon the high specific surface area percentages and hydrophilic functional groups, including such hydroxyl and carboxyl groups, linked to its surface. Additional adsorption sites, such as voids and flaws, are created when PVA is mixed into GF layers. They can be formed at the interface between the two nanomaterials, which helps to increase humidity sensing. Moreover, GF has a low electrical resistance and a high concentration of charge carrier. It can act as an anchor to enhance electron transport in metallic nanoparticles, which helps to modify PVA. When the sensor is exposed to varying humidity levels in this study, it easily interacts with the water vapor, causing the dielectric constant to fluctuate. As per the findings of the research, GF nanostructures without PVA have a lack of stability and reproducibility, which is a stumbling barrier for sensor applications. PVA will absorb or desorb water molecules in a humid atmosphere, resulting in a change in the dielectric constant. The dielectric constant will increase as the humidity level rises, electrical response, i.e., capacitance and impedance, will change as the dielectric constant changes. When humidity rises, there will be more hydrogen bonding between water vapor and the OH groups in PVA. The quantity of HThe suggested sensor was evaluated for various applications depending on its outstanding sensing capability, including non-contact humidity sensing of the fingertip. Furthermore, we put our sample to the test for human healthcare monitoring applications by introducing it to human respiration to detect water vapors. Our sensor quickly responded to and detected minor %RH fluctuations. In summary, this paper proposed a highly sensitive, fast, and stable humidity sensor based on 2D graphene nanoflakes, with PVA/GF nanocomposite film as the sensing layer. The spray-coated GF solution has a high electrical conductivity and homogeneous dispersion. The suggested humidity detecting device showed a humidity sensing range of 40–90% RH, with the response and recovery periods of 2 s and 3.5 s, respectively, at room temperature. The developed humidity sensor exhibited a capacitance sensitivity response of 29 nF/%RH at 10 kHz with a good linearity and reproducibility. Our proposed device also exhibited different applications, such as contactless skin humidity monitoring, due to its superior sensing performance characteristics. Similarly, we tested our sensor in an open environment and found it to be stable. The humidity detecting performance of the constructed sensor was outstanding. Based on these findings, we anticipate that humidity sensors based on PVA/GF nanocomposite will have a bright future in the field of real-time humidity detecting electronic devices.


Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed. Introns, or intervening sequences, are segments that are transcribed but removed from a transcript before translation can proceed. There are several categories of introns, such as tRNA introns, nuclear spliceosomal introns, and Group I and Group II introns . IntronsOrganellar genomes can be quite variable in size due to the absence and presence of introns, however, little is known about the mechanisms that promote intron gain and loss and why in some instances, the organellar genomes are devoid of introns, or in extreme examples where introns comprise most of the nucleotides that makeup the genome ,9,10. OrOrganellar introns are mainly classified as self-splicing introns that are either Group I and II type introns, based on differences in their splicing pathways and RNA secondary structural features. Sequence conservation among Group I or Group II introns is very low, but conservation among these elements can be found at the structural level ,17,18,19Group I and II introns at the RNA level assume RNA folds that generate the active sites needed for the splicing reactions to be initiated A,B. Grou2+ ions that permits the 2′OH Group on the budged adenine to destabilize the phosphodiester bond (PDE) that links the upstream exon with the intron . Ad. Ad16]. ) intron ,258. “Zoox3 gene . With thox3 gene ,259.Ophiostoma ips revealed two examples of complex introns: cytochrome b (cob I4) and the cytochrome oxidase III (cox3 I2) introns. The cox3 I2 intron is composed of two Group IA1 type intron modules in a possible tandem arrangement, and most likely is excised as a composite intron (‘zombie’ twintron). The complex cob I4 intron is the first report of a trintron, which is a complex intron composed of three intron modules. Two Group I introns are in tandem arrangement with the first intron module being interrupted by a Group II intron module. Based on in silico predictions this complex intron could splice by a ratchet-type mechanisms, whereby the upstream Group I intron module splices first followed by the splicing of the downstream Group I intron module. The Group II intron module interrupts the ORF of the first intron module and the Group II module is assumed to splice independently of the other intron modules [Characterization of the mitogenome for the fungus  modules .Monomorphina aenigmatica [Atypical Group II introns and twintrons have been identified in the plastid genome of the freshwater euglenoid igmatica . Throughigmatica . To be aChaetomium thermophilum var. thermophilum, in which the splicing of the internal Group II intron reconstitutes a HE ORF of the external (resident) Group I intron, facilitating its expression which, in turn, contributes to the mobility of the twintron to cognate intron-less rns genes [E. coli; within E. coli, the expressions of the intron encoded protein could be manipulated by modulation the splicing of the internal Group II intron module by manipulating Mg2+ concentrations in the media [E. coli.Twintron/complex introns offer new ribozyme scaffolds that could be engineered where the expression of intron encoded ORFs can be regulated by the splicing of nested ribozyme type components . An examns genes . It was he media . EssentiThe study of complex introns is interesting from an evolutionary point of view, with these configurations evolving independently multiple times involving different categories of introns, to generate complexity. These complex ribozymes my evolve into platform of alternative splicing or as regulatory elements that can modulate the expression of the host gene ,263. TheExploring organellar genomes and their intron complements is important as these introns can impact genome sizes, genome architecture, and organellar genome diversity. Organellar introns have the potential to serve as regulatory elements that can impact gene expression and, in some instances, introns are associated with a phenotype ,269. MutTetrahymena thermophila can function in RNA repair by replacing mutated regions of mRNA with corrected sequences and, thus, contribute to therapeutic strategies [Polymorphism in mitochondrial Group I introns have been linked to drug susceptibility in fungal pathogens and are being explored as important markers in clinical research . Trans-srategies . Group Irategies ). Organerategies ,272. Forrategies ,274. In rategies ,274; revrategies ,276), corategies ,277,278.Group I and II introns, complex introns, and trans-splicing introns could be considered examples of constructive neutral evolution where complex systems evolved by non-adaptive mechanisms  ,280. Pos


In recent years, two‐dimensional (2D) materials have attracted a lot of research interest as they exhibit several fascinating properties. However, outside of 2D materials derived from van der Waals layered bulk materials only a few other such materials are realized, and it remains difficult to confirm their 2D freestanding structure. Despite that, many metals are predicted to exist as 2D systems. In this review, the authors summarize the recent progress made in the synthesis and characterization of these 2D metals, so called metallenes, and their oxide forms, metallene oxides as free standing 2D structures formed in situ through the use of transmission electron microscopy (TEM) and scanning TEM (STEM) to synthesize these materials. Two primary approaches for forming freestanding monoatomic metallic membranes are identified. In the first, graphene pores as a means to suspend the metallene or metallene oxide and in the second, electron‐beam sputtering for the selective etching of metal alloys or thick complex initial materials is employed to obtain freestanding single‐atom‐thick 2D metal. The data show a growing number of 2D metals/metallenes and 2D metal/ metallene oxides having been confirmed and point to a bright future for further discoveries of these 2D materials. In this review, the authors summarize the recent progress made in the synthesis and characterization of these 2D metals, so called 2D metals/metallenes, and their oxide forms, 2D metals/metallene oxides as free standing 2D structures formed in situ through the use of TEM and STEM to synthesize these materials. Hence, they have been studied intensively in the last decade. 2D materials such as graphene, h‐BN, and transition metal dichalcogenides (TMD), which are used widely, are known to exhibit strong intralayer covalent bonding and weak interlayer van der Waals bonding. Thus, they can be exfoliated with ease to form atom‐thick layers. On the other hand, metals with metallic bonding (isotropic) preferentially exhibit 3D close‐packed structures, which presumably cannot be exfoliated to form 2D structures. Therefore, currently, research on 2D materials is mostly limited to van der Waals structures.2D materials can exhibit exceptional surface‐related properties because their atomic‐scale thickness leads to surface and quantum confinement effects, such as the quantum Hall effect,1, 2 and few‐layer materials. For instance, membranes of potassium on graphene, single‐atom‐thick layers of Pb and In on Si(111), Hf layers on Ir(111), Rh nanosheets on poly(vinylpyrrolidone), and Ga layers on different substrates have been reported. Hence, the term “metallene” is being used to refer to metals, which do not show a layered structure in the bulk form and can exist as a 2D layered structure. An example would be silicene (the 2D form of silicon). Similarly, tin can yield stanene, while germanene is the 2D form of germanium. In addition, antimonene and plumbene have also been reported. As 2D materials, 2D metals/metallenes are assumed to be sufficiently stable in their freestanding form . However, this has not yet been demonstrated experimentally. The first freestanding iron membrane with single‐atom thickness was successfully fabricated by Rümmeli et al., in 2014, using an electron beam (e‐beam). This confirmed the existence of 2D metals/metallenes as a new class of 2D materials. In keeping with previous approaches, such as the epitaxial growth of a 2D metals/metallene using a small‐lattice‐mismatch substrate, Rümmeli et al. used the e‐beam of a TEM system to drive the formation of Fe surface atoms and nanoparticles (NPs) into Fe 2D membranes suspended in graphene pores. This increased research interest in freestanding 2D metals/metallenes as well as the possibility using e‐beams to form novel 2D materials.Research on 2D metals was initiated with reports of the existence of single6 and  Ag, and Cu. In a systematic investigative study by Nevalaita et al., the 2D structures of 45 different metals were analyzed through density functional theory (DFT) simulations. The calculations showed that most of the 2D metals are more stable in the hexagonal‐ and honeycomb‐structured forms than in the square‐lattice‐structured form. The cohesive energy plays an important role in the formation of 2D metals. A stable 2D metal must exhibit a balance between the 2D cohesion, which must be sufficiently high, and the 3D bulk cohesion, which must be sufficiently low. The 2D structure will be unstable if the 2D cohesion is too low, while a 3D structure is formed if the 3D bulk cohesion is too high. Hence, both theoretical and early experimental studies suggest the existence of a new class of 2D elemental metals in the form of freestanding structures.Inspired by the discovery of Rümmeli et al., there have been several theoretical efforts to predict the existence of other freestanding 2D metals/metallenes, such as those of Au,20 Ag,1.2 Various types of interactions can occur during TEM when the energetic electrons penetrate the test specimen. The incident electrons lose their energy (or experience a change in their momentum), and this energy is transferred to the specimen atoms, resulting either in the heating of the specimen or the breakage and rearrangement of the chemical bonds between its atoms. On the other hand, a few electrons can travel through the specimen without any (or very little) loss in energy. These two events can be categorized as corresponding to two different types of electron interactions, namely, inelastic and elastic interactions.As discussed in the previous section, TEM is not merely a simple tool for characterizing 2D materials. During observations, its e‐beam also interacts with the test specimen. For instance, in the case of Fe NPs, it can drive the NPs to form a monoatomic Fe membrane. This is a typical example highlighting the power and versatility of advanced high‐resolution TEM (HRTEM) systems in materials science, physics, and chemistry. The energetic electrons can not only yield morphologic, compositional, and crystallographic information regarding the test specimen but can also drive in‐situ reactions with the specimen  at the atomic level.30 Var The signals produced by inelastic interactions are mainly employed in analytical electron microscopy methods, such as electron energy loss spectroscopy (EELS) and energy‐dispersion X‐ray spectroscopy. In addition, such inelastic interactions may also cause various effects, such as specimen heating, ionization‐induced damage (radiolysis), and hydrocarbon contamination. In the case of elastic interactions, the electrons do not transfer any energy (or only a negligible amount of energy) to the specimen. Such electrons are used in the case of TEM and electron diffraction analysis methods. In addition, these elastically scattering electrons may induce various phenomena such as electrostatic charging, atomic displacement, and e‐beam sputtering. These e‐beam‐induced phenomena can be exploited for the in‐situ TEM analysis of 2D materials.In the case of inelastic interactions, the incident electrons transfer a certain amount of energy to the atomic electrons in the specimen. This transferred energy can generate different signals, such as Auger or secondary electrons, X‐rays, plasmons, and phonons.32 The1.3 such as those related to their growth, etching, and reduction/oxidation. During such in‐situ (S)TEM studies, the e‐beam plays the dual role of inducing the reactions while simultaneously recording them. This is extremely important because the synthesis of 2D materials requires a comprehensive understanding of their characteristics, and in‐situ observations of their growth process at the atomic level can provide this information. In other words, (S)TEM can elucidate the growth mechanism of 2D materials. In addition, while in the case of other methods for synthesizing 2D metals/metal oxides, it may be challenging to demonstrate the freestanding structure of the synthesized material; this difficulty can be overcome using in‐situ (S)TEM TEM and highlight the importance of in situ (S)TEM in the development of freestanding 2D materials.High‐resolution (scanning) TEM (HR(S)TEM) with advanced upgraded spherical aberration (Cs) correction and reduced chromatic aberration (particularly for low acceleration voltages) has emerged as an ideal technique for studying the lattice structure of advanced materials and, in particular, 2D materials, which become reactive under an e‐beam. The subatomic spatial resolution of advanced HR(S)TEM allows one to visualize the atomic structure and bonding configuration of materials and track the movement of their atoms with high temporal resolution. Furthermore, the elastic and inelastic interactions of the electrons with the specimen, which may induce radiolysis, hydrocarbon contamination, atomic displacement or sputtering, bonding rearrangement, and crystallization, can be usefully exploited for the in‐situ (S)TEM e‐beam‐driven chemical reaction studies of (e‐beam‐reactive) 2D materials,35 suc2 at its edges instead of on‐top adsorption; this results in exothermic interactions (in the suitable direction) between the atoms of a metal layer formed on it and the pore edges of graphene, which, in turn, help stabilize the 2D membranes formed within the graphene pores. Owing to this phenomenon of stabilization of 2D metal/metal oxide membranes, the use of graphene pores as growth templates is an extremely promising approach for the growth of freestanding 2D metal/metal oxide membranes.In addition to exhibiting several desirable properties, graphene shows preferential in‐plane adsorption46 at 2.1  during the transfer process or intentionally using a condensed electron beam. Under e‐beam irradiation, the Fe nanocrystals located near the graphene pores started to collapse into a disordered structure. The Fe atoms that were freely mobile on the graphene substrate under the e‐beam were attracted by the graphene hole edges and moved toward the holes, recrystallizing into a square lattice and forming a monolayer 2D Fe membrane suspended within the graphene pores. The results of DFT calculations were consistent with this finding, in that they also indicated that the formation of a single‐layer 2D square lattice Fe membrane is energetically favored instead of the other possible 2D structures, such as tetragonal and hexagonal ones. The experimentally measured lattice constant for this membrane (2.65 ± 0.05 Å) was larger than that of the (200) face‐centered cubic phase or the (110) body‐centered cubic (bcc) phase of 3D bulk Fe. This increase in the lattice constant (near the membrane center) is attributable to the lattice alignment and mismatch between the graphene substrate and the 2D Fe monolayer, which results in a strain. In addition, the calculations also showed that the magnetic moment of single‐atom‐thick Fe membranes (3.08 μB) is significantly higher than that for bulk bcc Fe (2.1 μB). The largest stable membrane as per the experimental data and DFT calculation results was 10–12 atoms in width.As discussed previously, a single‐atom‐thick 2D Fe membrane was the first freestanding 2D metals/metallene grown in graphene pores using an e‐beam19  crystals. ii) O was then preferentially sputtered from the oxide nanocrystals under continued e‐beam irradiation, leading to the formation of a freestanding 2D single‐atom‐thick Cr membrane, which was suspended within the graphene pores. The local EELS profiles were measured to confirm that the membrane comprised pure Cr. In addition, the EELS spectra of the Cr‐L2,3 edges from different Cr based forms  were studied further. The Cr‐L3/L2 white‐line intensity ratio from the 2D Cr membrane (<1) was found to be significantly smaller than that compared to other structures reflecting the different bonding configuration of the 2D single atom thick Cr membrane as compared to the other Cr forms. DFT calculations indicated that the ground state of the 2D Cr membrane was antiferromagnetic, making it suitable for spintronic applications and further highlighting the existence of a new class of 2D metals/metallenes.In another experimental study, Ta et al. reported the in‐situ synthesis of a freestanding single‐atom‐thick 2D metals/metallene using an e‐beam.36 A s2.2 They observed small Pt clusters embedded within graphene pores. Under e‐beam irradiation, these clusters gradually transformed into a planar structure, as shown in Figure2D metals do not always form a crystalline freestanding membrane under e‐beam irradiation. Freestanding 2D metal clusters have also been observed. For example, 2D metal nanoclusters suspended within graphene holes were first reported by Dong et al.37 The also reported observing not a 2D metals/metallene  but novel 2D single‐atom‐thick Sn membranes suspended within graphene pores, as shown in Figure ) under irradiation from the e‐beam (80 kV) of an LVACTEM system. This resulted in novel single‐atom thick 2D planar clusters/membranes with sizes of 1–8 atoms embedded within the graphene pores. A star‐like or close‐packed structural configuration was observed in the case of the patches of three or more atoms.Yang et al.38 alsThe team also employed DFT studies to look at the cluster configurations and energetics. The results deviated from experimental observations for membranes larger than five atoms and this was attributed to the interfacial forces between the graphene pore edges and Sn atoms not having been including in the calculations. However, in initial interfacial study looking at the adsorption energetics of a Sn atom adsorbed on graphene or within a C vacancy was conducted and the study confirmed that pores can stabilize Sn atoms through interactions with the graphene pore edges. Under e‐beam irradiation, the shape of the underlying graphene pores change, leading to the dynamic transformation of the Au nanoclusters into freestanding 2D membranes, which subsequently turn into freestanding 2D nanoribbon with a ribbon width of 4–7 atoms. The Au ribbon structures match that of the top monolayer of bulk Au (111), viz, a hexagonal close‐packed lattice matching very well with previous theoretical predictions. Moreover, the theoretical predictions suggest 2D single‐atom‐thick Au structures are metallic and diamagnetic with metallic bonds holding the gold atoms together in the 2D plane. Experimentally, these freestanding 2D Au nanoribbons showed good stability under irradiation with an 80‐kV e‐beam.In a further study, freestanding single‐atom‐thick 2D Au nanoribbons, which are another form of freestanding single‐atom‐thick 2D metal structures, were reported by Zhao et al.39 Und2.3 reported the e‐beam‐driven in‐situ formation of freestanding graphene‐like ZnO (g‐ZnO) mono‐ and bilayer membranes in graphene pores from ZnO NPs using an LVACTEM system at 80 kV. Theoretical calculations and experimental studies have shown that the bulk structure of ZnO (wurtzite) with a thickness of a few atoms can transform into a supported graphene‐like planar structure. However, freestanding 2D membranes of g‐ZnO had not been realized until first reported by Ta et al. The lattice constant of the transformed g‐ZnO membranes from the membrane center towards the graphene–membrane interface at the outer membrane edges was higher by 1.6% (a = 3.303 Å). This increase can be attributed to the lattice mismatch between the graphene substrate and the 2D g‐ZnO membrane. ZnO was obtained by the thermal decomposition of Zn acac under high vacuum, which led to the formation of ZnO (wurtzite) NPs over the graphene substrate. Under e‐beam irradiation, the small ZnO NPs (outer diameter of ≈1–2 nm) formed on the graphene substrate were highly reactive, and their crystal structure changed readily to an amorphous one, in which the Zn and O atoms were highly mobile. Under extended irradiation, the dynamic Zn and O atoms recrystallized, forming a single‐layer graphene‐like structure  were attached to the step edges of multilayered graphene flakes (Panel II(a)). Driven by a scanning focused e‐beam (60 kV) used in the STEM mode, these clusters moved around the graphene edges, and their morphology changed continuously. At the critical point, they transformed into a crystalline single‐layer structure, with the Cu atoms forming a square sublattice. The experimentally measured value of the lattice constant, dCu‐Cu, was 2.69 ± 0.05 Å and in agreement with the results of the DFT calculations for CuO (dCu‐Cu of 2.67 Å). The small copper oxide clusters close to the graphene holes also transformed into a monolayer freestanding membrane under low‐dose‐rate e‐beam irradiation. A suspended monolayer with a square Cu sublattice is displayed in the STEM‐annular dark‐field (ADF) image in Panel II(b)), and the FFT of the image is shown in Panel II(c). The experimentally measured dCu‐Cu value is 2.65 Å, which is also close to the DFT‐calculated value of 2.7 Å for freestanding monolayer CuO in graphene pores (Panel II(d)).Figure 40 A s3 ion‐beam irradiation, laser irradiation, plasma treatment, scanning electron microscopy (SEM), and chemical and thermal treatments. Herein, we focus on approaches that use TEM in the conventional and scanning (STEM) modes to etch or thin 2D materials in a layer‐by‐layer manner to form atomically thin regions as well as novel materials.A strong understanding of the underlying theory and precision in the manipulation of materials are essential for achieving the desired control over material properties. The manipulation and engineering of materials at the atomic scale can be achieved through different methods, such as e‐beam irradiation,53 ion However, e‐beam irradiation, when used with precision in an assisted manner, can be employed advantageously to engineer materials in a controllable manner. For instance, material engineering can be performed with precision using a TEM system. This is because TEM allows for the simultaneous high‐resolution imaging and monitoring of the in situ dynamic transformations that occur during the e‐beam irradiation process. The real‐time monitoring of the material transformations also allows for the irradiation parameters to be changed. Thus, the desired results can be achieved with a high degree of control.The thinning of materials through e‐beam irradiation can occur through elastic and inelastic scattering. These scattering processes are governed by different factors, such as the nature of the material in question and the energy/dose of the incident e‐beam. Furthermore, these processes can give rise to different phenomena, such as charging, atom displacement, sputtering, heating, and radiolysis. These phenomena may induce damage in the irradiated material.66 How showed that by varying the electron beam acceleration voltage it is possible to controllably cut and thin graphene monolayers using a focused electron beam. Figure reported that they were able to thin a few layers of tungsten disulfide (WS2) using TEM and STEM. They demonstrated that an e‐beam produced using an acceleration voltage of 200 kV caused the local thinning of WS2. Furthermore, the changes in the structure could be monitored, yielding insights into the development of new techniques for the nanoscale modification of 2D WS2 as depicted in different snapshots of Figure The aim of such manipulations is to ultimately fabricate materials with specific properties that can be used in next‐generation technologies. For example, Dyck et al.66 sho To control the damage caused by the irradiated electrons and consequently control the number of phosphorene layers with precision, the authors used graphene as a protective layer for the multilayered BP. The graphene layer acted as a barrier to prevent radiolysis damage and increase the stability of the BP. The authors also tested different configurations of the graphene/BP heterostructure, namely, graphene on the top of the BP, graphene at the bottom of the BP, and graphene as an encapsulating layer for the BP. The presence of graphene on only one side (top or bottom) of the BP layers allowed for controllable thinning under e‐beam irradiation. On the other hand, in the case of the encapsulated BP configuration, the amorphization of the BP layers was observed. The higher stability of the graphene/BP heterostructure allowed the etching mechanisms in play during the e‐beam irradiation process to be investigated. Thus, it was shown that graphene can be used to aid the e‐beam thinning of 2D materials as well as for detailed investigations of the defects of such materials and their effects on the material properties.Mono‐ and bilayer phosphorene could be produced through the layer‐by‐layer etching of black phosphorus (BP) using an e‐beam as shown in Figure 68 To  demonstrated that it is possible to controllably remove S and Se atoms from 2D tin dichalcogenides (SnS2 and SnSe2) using an e‐beam, .Sutter et al.69 dem, Figure  Panel IV described the etching of MoS2 monolayers with zigzag edges. To accomplish this, they annealed the MoS2 sample at 800 °C with a blanked electron beam and then tracked the sulfur vacancies caused by electron irradiation with a 60 kV beam. This atom‐by‐atom approach also allowed the formation of MoS2 nanoribbons atom‐by‐atom. Tai el al. also modified the structure of MoS2 layers through the use of an electron beam and heating, leading to the etching and sculpting of multiple MoS2 structures in the electron‐irradiated area. The in situ work showed the possibility to etch, fold, and scroll MoS2 monolayers. Moreover, their results demonstrated the possibility to create hybrid lateral heterojunctions composed of 1H, 2H, 3R, 3R’, and TZ stacking configurations.Chen et al.70 des used aberration‐corrected STEM to etch a MoS2/WS2 heterostructure in a layer‐by‐layer manner until the formation of nanopores that were arranged in different arrays across the 2D lattice. The thinning process was performed at room temperature using a TEM system at 80 kV. By varying the focus of the e‐beam in the STEM mode, they observed that the MoS2 layer was etched first. This was followed by the ablation of the WS2 layer, resulting in the formation of pores/defects in the irradiated region. The size of these pores/defects could be controllable to 0.6 nm.Das et al.72 use Continuous electron irradiation resulted in the formation of ferromagnetic nanoribbons with widths of 0.65–0.9 nm. Figure2, R3, and R4) of the Au membranes were calculated from the high‐resolution TEM images, as shown in Figure In another study, atomically thin freestanding Au membranes were fabricated through the local dealloying of Au and Ag atoms using e‐beam irradiation.41 Con subjected 2D Mo2Ti2C3 monolayers to e‐beam irradiation in the TEM mode at 300 kV. The freestanding monolayers were irradiated for 6 min, and the thinning process was monitored. EELS showed that the irradiated areas of the Mo2Ti2C3 monolayers remained stable for the first 2 min. However, this was followed by the formation of sites with a high Mo content and regions with a high C content. These sites and regions evolved during the remaining 4 min of irradiation. In the first case, e‐beam irradiation led to the constant removal of Mo atoms until a network of Mo nanoribbons was formed. Continuous irradiation meant that the Mo nanoribbons were thinned continuously until a single‐atom chain formed and ruptured. In the second case, Ti and Mo were removed gradually. The thinning process resulted in the formation of monoatomic N‐doped graphene areas, as shown in Figure +) during the e‐beam irradiation process. The presence of TBA+, in turn, can be ascribed to the delamination process used for forming the Mo2Ti2C3 monolayers. Figure 2Ti2C3 monolayers. Figure The thinning of 2D materials can result in the formation of novel materials such as N‐doped graphene and Mo nanoribbons. Mendes et al.42 sub were able to form Mo5S4 nanoribbons by thinning MoS2 monolayers through e‐beam irradiation. They found that the irradiation of the MoS2 monolayers using a TEM system at 80 kV caused the formation of defects, which evolved into holes with a diameter of 3–6 nm. Under continuous irradiation, the connecting regions between the holes narrowed to form nanoribbons with a width of 0.35 nm and bandgap of 0.77 eV. The authors claimed that, given the ease of controlling the formation of these nanoribbons and their high stability, they can be used in various functional devices.Similarly, Liu et al.73 wer used an aberration‐corrected TEM system operating at voltages higher than 80 kV to preferentially remove C from 2D α‐Mo2C crystals. The irradiation process led to the formation of thin rectangular Mo structures with lattice parameters of 0.233 and 0.167 nm, as shown in Figure α‐Mo2C crystals by tuning the energy of the incident e‐beam.The development of controllable methods for tuning the properties of 2D materials by either reducing their size or creating new nanostructured materials is pivotal to the realization of next‐generation functional devices. Si et al.43 use2 using a focused beam in the STEM mode. The weak nature of the Mo–Se bond makes the formation of 2D Mo membranes embedded within the MoSe2 lattice easy. Figure 2 lattice, leading to the formation of 2D Mo membranes in selected regions of the initial MoSe2 crystal, as indicated by the green‐shaded areas in Figure 2 film with a lattice parameter of 0.33 nm. For comparison, Figure 2D Mo membranes have also been fabricated by selectively removing Se atoms from monolayer MoSeTEM mode.44 The4There are several important parameters in the fabrication of 2D metal/metallene or 2D metal/ metallene oxides using electron beams as the driving mechanism for their fabrication. Typically these include the electron acceleration voltage, the electron beam dose  etc. These key parameters of course vary depending on the specimen composition and they are summarized in the tables presented below. The tables are arranged in two fabrication approaches, namely, growth in supports such as a graphene pore or hole and layer thinning through electron beam irradiation.TableIn the case of forming 2D membranes  in a graphene pore Table1, mostTableThe main parameters governing the thinning of 2D nanomaterials to form freestanding single atom thick 2D metals/metallenes and 2D metals/metallenes oxides presented in this review using an electron beam are provided in Table2), making it harder and in many cases preventing a direct real‐time observation of the thinning process.5 has been increasing steadily in the last few years, it remains a significant challenge to even observe these materials experimentally. For example, only a limited number of freestanding 2D metals/metallenes have been synthesized so far  to obtain freestanding single‐atom‐thick 2D metal membranes. The data show a growing number of 2D metals/metallenes and 2D metals/metallene oxides having been confirmed and point to a bright future for further discoveries of these 2D materials.Novel 2D metals and metal oxides show unique properties and hence have significant potential for use in a range of applications, making them both interesting and challenging research subjects. Several approaches have been reported for synthesizing these materials and confirming their existence. However, given the recent advancements in reducing chromatic and spherical aberrations, which allow for the use of low acceleration voltages while maintaining a high atomic resolution—this is particularly important with respect to low‐dimensional materials—HRTEM has emerged as an ideal approach for studying 2D metals/metallenes and 2D metals/metallene oxides and confirming their existence. During HRTEM, the irradiation of the e‐beam leads to in‐situ reactions involving the test specimen, resulting in novel 2D materials. In this review, we summarized the recent achievements made with regard to the use of in situ (S)TEM for studying the freestanding configurations of 2D metals/metal oxides, which has remained challenging till now. With respect to in situ (S)TEM, there are two primary approaches for forming freestanding monoatomic membranes. The first is using graphene pores as templates, so that the e‐beam causes the metal/metal oxide atoms to fill the graphene pores and crystallize and thus form a freestanding 2D crystal membrane. The second is e‐beam sputtering or the selective etching of metal alloys  or thick complex initial materials (e.g., MoThe authors declare no conflict of interest.