Volumetric‐modulated arc therapy (VMAT) has been shown to be able to deliver plans equivalent to intensity‐modulated radiation therapy (IMRT) in a fraction of the treatment time. This improvement is important for patient immobilization/ localization compliance due to comfort and treatment duration, as well as patient throughput. Previous authors have suggested commissioning methods for this modality. Here, we extend the methods reported for the Varian RapidArc system  to the Elekta linear accelerator, using custom files built using the Elekta iComCAT software. We also extend the method reported for VMAT commissioning of the Elekta accelerator by verifying maximum values of parameters  speed, and backup jaw speed), investigating: 1) beam profiles as a function of dose rate during an arc, 2) over/under dosing due to MLC reversals, and 3) over/under dosing at changing dose rate junctions. Equations for construction of the iComCAT files are given. Results indicate that the beam profile for lower dose rates varies less than 3% from that of the maximum dose rate, with no difference during an arc. The gantry, MLC, and backup jaw maximum speed are internally consistent. The monitor unit chamber is stable over the MUs and gantry movement conditions expected. MLC movement and position during VMAT delivery are within IMRT tolerances. Dose rate, gantry speed, and MLC speed are accurately controlled. Over/under dosing at junctions of MLC reversals or dose rate changes are within clinical acceptability.PACS numbers: 87.55.de, 87.55.Qr, 87.56.bd A shorter treatment time is a significant improvement for both patient compliance issues, due to confining immobilization and respiratory control devices, and patient throughput. While IMRT arc therapy is not new,(40cm×40cm) is due to delivery and planning systems that can now modulate and monitor gantry speed, dose rate, and MLC position simultaneously. To verify that the linear accelerator (linac) can correctly deliver a VMAT treatment, Ling et al.Volumetric‐modulated arc therapy (VMAT) is being used to deliver intensity‐modulated radiation therapy (IMRT) treatments with shorter treatment duration for each fraction and, in some cases, a better dosimetric plan.,,, A The accelerator tested for this project was an Elekta Infinity with the MLCi2. The MLC has 40 leaf pairs, each with a width of 1 cm at isocenter, a minimum gap of 0.5 cm at isocenter; the MLC is interdigitation‐capable . Both 6 and 10 MV will be used for VMAT delivery. Tests with no dose rate (DR) considerations were carried out for 6 MV only. We constructed VMAT files controlling each component of the delivery using the iCom Customer Acceptance Test (iComCAT) version 1.0.0.13 . Care must be exercised using this program in accordance with the caution noted: the software “lacks safeguards normally present in a program intended to prepare and deliver clinical prescriptions”.http://imagej.nih.gov).The electronic portal imaging device (EPID) was used for the MLC positioning (Picket Fence) and other tests. Imager sag was characterized by taking images using the EPID of open fields with an open‐air graticule  in place. The image analysis software used for the central BB locations, as well as other EPID analysis, was ImageJ (available at The MapCHECK2 was used for dosimetric measurements. It was mounted to the gantry using the Isocentric Mounting Fixture (IMF)  with a 3 cm buildup plate to give an effective depth of 5 cm (the device has 2 cm of inherent buildup). Diodes used for the measurements were in areas designed to achieve uniform dose, so geometric accuracy is not as critical as the EPID, but nevertheless needed to be quantified. This was measured by mounting the device to the IMF without buildup and measuring the distance of the light‐projected crosshair to the center mark of the device. Since the sag might be different with the 3 cm buildup plate, the plate was placed on the IMF at each gantry position without obscuring the crosshair.For brevity and consistency with others, we use the following in equations and when identifying particular values of each parameter:θ=gantryangle; Δθ/Δt=gantryspeed; x=positionofanMLCleaf; Δx/Δt=leafspeed; MU=doseinmonitorunits; ΔMU/Δt=doserate; Δθ/ΔMU=gantryangletraversalperMU; max=maximum; min=minimum.Also for brevity and reading ease, we use additional abbreviations for dose rate (DR) and gantry speed (GS).Dosimetric tests for general linac performance were carried out by positioning an ionization chamber with a buildup cap at isocenter. Charge was measured using an electrometer with high‐charge collection capability. These measurements are an extension of the work by Bedford and Warrington,10cm×10cm field with the ion chamber placed at 100 cm source‐to‐axis distance for both 6 and 10 MV beam. Monitor unit values were 36 and 1000 MUs with DR values of (ΔMU/Δt)min and (ΔMU/Δt)max, respectively. The 36 MU value corresponds to (Δθ/ΔMU)min. Comparisons were made between static and arc fields, and linearity between the two dose levels was calculated.The monitor unit chamber stability was tested over the range of expected VMAT MUs for both static and 360° arc fields using a 23cm×10cm field with a dynamic moving gap of 1 cm with the gantry at cardinal angles and a 180° arc  and measured using a Farmer‐type ionization chamber with a buildup cap with the cylinder barrel oriented perpendicular to the MLC motion direction. The collimator angle was oriented to have the maximum gravity effect on the MLC movement .  Comparisons between each measurement with the average static measurement were carried out.Gravity effects on a dynamic treatment were investigated by constructing a irection.,,8 25cm×25cm field at a vertical and horizontal angle, as well as during a 358° arc, which was thought to be sufficient to identify any problems. The absolute MUs delivered for the arc case with different DRs were changed for each arc to maintain a gantry speed between 4° and 4.5°/sec, except for the lowest dose rate which had a gantry speed of about 3°/sec (necessary so the maximum gantry speed was not exceeded). The depth of measurement was 5 cm equivalent for both 6 and 10 MV. The dose rates we show are nominal to compare with Bedford and Warrington;(ΔMU/Δt)max/32, which we do see occasionally during VMAT treatments. Note that the beam profile measurements for each DR for the vertical gantry were used for normalization of subsequent tests (see below). An EPID or film can also be used for these measurements, but the 2D diode array is sufficient in the authors' opinions, especially considering the convenience and ease‐of‐use. We encountered pixilation effects with the EPID for several of the tests in the low DR region that made the 2D diode array more attractive.Bedford and Warrington25cm×25cm; the EPID was used to acquire the dosimetric‐positional image.Dynamic Picket Fence patternsFor these tests, a Picket Fence with a moving gap of 5 mm was used, except at desired positions a 1 mm strip was enhanced by advancing the leading MLC 1 mm and pausing the trailing MLC over the 1 mm strip; but either or both MLCs are moving continuously. The collimator angle was oriented to have the maximum gravity effect on the MLC movement . The Picket Fence test was run at the cardinal angles and a 358° arc. EPID image data was captured using ImageJ and imported into a spreadsheet to quantitatively analyze the Picket positions.As with Ling et al.,For the Elekta linac, maximum values for gantry speed, MLC speed, backup jaw speed, and collimator rotation speed are input into the software and are readily displayed. TPSs currently do not allow for dynamic collimator rotation, so this was not tested. Rather than timing these values absolutely, which introduces subjectivity due to ramp‐up speed, we chose to verify these for internal consistency using the following tests.DRmax to determine the GS and MLC speed. To determine the accuracy of the displayed DRmax, 500 MUs were delivered for both 6 and 10 MV and timed with a stop watch. The time needed for the average DRmax displayed was compared with the measurement.Presently, ΔMU/Δt for our Elekta accelerator is binned as multiples of half of the maximum DR, which are displayed. We used the 10cm×10cm field using a DMLC with an 8 mm gap  in the iComCat software. The MUs for this field are calculated using Eq. A maximum MLC speed of 2 cm/sec is recommended by the vendor and was entered in the controlling software. This was tested for internal accuracy by constructing a (xf−xi) is the final and initial MLC position, which was 10 cm for our test.where (Δx/Δt)max. Similarly if the displayed DR is half‐maximum, the maximum MLC speed is less than the nominal speed; the MUs can be incrementally increased for the field until they run at (ΔMU/Δt)max, at which point the MLCs will be at the maximum speed. Fractional MU entry is allowed, so MLC speed within 0.1 cm can be calculated.The calculated MUs and other field parameters were input into an iComCat file and executed. If the displayed DR is maximum, the MLC speed may be greater than 2 cm/sec, and the MUs can be incrementally decreased until the DR drops to half‐maximum. The actual MLC speed can then be calculated by solving Eq. The maximum backup jaw speed in the controlling software was 1.5 cm/sec. The same method as given in C.1.1 for the MLC speed was used to verify internal consistency of this value.The gantry speed given by the vendor was 6.0°/sec which was input into the controlling software. The MUs for the verification measurement were calculated using:(θf−θi) are the final and initial gantry positions, which was 180° for our test, and (Δθ/Δt)max was the nominal maximum given by the vendor.where (Δθ/Δt)max.The parameters for the nominal gantry speed from Eq. (ΔMU/Δt)max/32:(Δθ/Δt)max/32; (ΔMU/Δt)max/16:(Δθ/Δt)max/16; (ΔMU/Δt)max/8:(Δθ/Δt)max/8; (ΔMU/Δt)max/4:(Δθ/Δt)max/4; (ΔMU/Δt)max/2:(Δθ/Δt)max/2; (ΔMU/Δt)max:(Δθ/Δt)max; 1.2(ΔMU/Δt)max:(Δθ/Δt)max; (ΔMU/Δt)max:1.2(Δθ/Δt)max. Note the last two strips have either the DR or GS greater than the maximum, to determine if the dose is still correctly delivered.To verify accurate DR and GS during VMAT delivery, a test with eight horizontal strip combinations of DR and GS was constructed where strips had DR:GS values of: The MUs delivered in each strip and moving between strips were constant to allow relative analysis, as suggested by Ling et al.These conditions were met by first calculating the time taken for the gantry arc between strips:ti→j= the time taken in moving between strips, and xi→j is the width of the strips. For this test, we chose (Δx/Δt)i→j=0.75(Δx/Δt)max.where Then:MUi→j are the MUs moving between strips. In selecting (ΔMU/Δt)i→j, the physical constraint ΔMU/Δθ≥0.1MU/∘ cannot be violated. This is verified using:where (ΔMU/Δt)i→j was chosen to be (ΔMU/Δt)max/8 so the DR ramp up/down would not be too extreme for the slower or faster DRs in each strip.Except for the strips with 1.2 times DR or GS, the total angle traversed is the same for each strip, and is calculated as:θi is the angle for the strip i; and where MUstripconstant were the MUs chosen to be significantly larger than the MUs delivered when moving from one strip to the next, but restricted to a value such that the sum of the gantry angles traversed for all strips and movement between strips is less than a complete arc, which can be calculated, but we solved it iteratively using a spreadsheet.where The strips were 3 cm wide with initial and ending segments of 0.5 cm. The measurement point was at the middle of each strip. The measurement device was the same used for the open beam profiles (above). The field sizes and measurement depth for the beam profiles and these measurements were the same, which allowed us to use the beam profiles to normalize the measurements here for the off‐axis ratio. The measurement points were at the middle of each strip, and the middle of the MLC leaf pair to minimize interleaf leakage considerations. To minimize the effect of a single aberrant diode measurement, 12 diodes in the strip were averaged . Each diode measurement was normalized using the open field with the same DR at the same detector position.(Δx/Δt)max/32:(ΔMU/Δt)max/32; (Δx/Δt)max/16:(ΔMU/Δt)max/16; (Δx/Δt)max/8:(ΔMU/Δt)max/8; (Δx/Δt)max/4:(ΔMU/Δt)max/4; (Δx/Δt)max/2:(ΔMU/Δt)max/2; (Δx/Δt)max:(ΔMU/Δt)max; 1.2(Δx/Δt)max:1.2(ΔMU/Δt)max. For the dose per strip to be constant for ease of analysis, the MUs were determined using:To test accurate control of MLC speed during VMAT delivery, an iComCAT file was constructed having a moving gap of 0.8 mm , constant GS, and strips having differing MLC speed and DR (MLC speed: DR): (xf−xi) are the final and initial MLC positions for each strip (3 cm width for each strip). The input GS was constant for this test, and was calculated using Eq. (ΔMU/Δt)i→j=(ΔMU/Δt)max/32. The actual deliverable gantry speed for the last strip with 1.2(Δx/Δt)max would be less by a factor of 1.2, since the MLC can't be driven faster than the maximum.where Strips of 1.5 cm width on the lateral sides of the field were also irradiated to the same dose so the field size would be the same as the open fields used in Section A.2.3 above, to enable normalization of the test field. Only 6 MV was used for the test, since it is an MLC speed test. The same measurement and analysis techniqueswere used as with the DR and GS test.As a cautionary note, we initially designed this test with the minimum gap of 0.5 cm, which gave poor results at the maximum MLC speed . Since these tests are outside of clinical mode which may have interrupted the treatment, this gap width using this software is not suggested when driving the MLC near maximum speed.(ΔMU/Δt)max/2 with differing numbers of traversals max. There are single reversals between the first four strips, and 4.5 reversals on the far right strip. The dose in each strip increases with the number of traversals. The EPID was used for the fluence measurement to obtain a continuous MLC movement profile and ImageJ was used for analysis. Individual pixel data at the junction changes was erratic, so the image data was smoothed manually by using a moving three‐pixel average . Smoothing was not thought to mask clinically significant effects occurring over 0.5 mm. The 1.0, 0.95, 0.90 (Δx/Δt)max measurements were normalized using the 0.18 (Δx/Δt)max measurement, which results in a relative analysis of uniformity in the ideal case of no over‐travel. The reference measurement corresponds to 0.36 cm/sec which was chosen by default using the maximum number of MUs for imaging (999 MU), and is assumed to have negligible over‐travel. The DR for the test (ΔMU/Δt)max/2 was chosen over the maximum to obtain the slower MLC speed for the reference measurement while providing adequate signal.Bedford and Warrington(ΔMU/Δt)max/32 to (ΔMU/Δt)max, sequentially by factors of 2, with the last two strips testing the extreme case of the dose increasing from (ΔMU/Δt)max/32 to (ΔMU/Δt)max. The MLC speed needed to be constant to investigate the DR alone; we were concerned that the maximum MLC speed may not give enough dose for an adequate signal, so (Δx/Δt)max/2 was used for the test. The fluence in each strip was in proportion to the DR for the strip. The gantry speed was constant for the test, with the speed calculated using Eq. (ΔMU/Δt)max/32 for (ΔMU/Δt)i→j and solving for (Δθ/Δt). As with the MLC rapid‐reversals test, the fluences were measured using the EPID to get a continuous profile, and the test measurement values were normalized for unity analysis using a reference measurement having all parameters the same except using a MLC speed of 0.25 cm/sec. Two measurements were made for both the test and reference irradiations, and were averaged pixel by pixel, again due to our concerns of the dose being too low for an adequate signal. Both energies used for VMAT, 6 MV and 10 MV, were tested.The effect of DR changes was investigated with other factors in previous sections using measurements at the middle of each strip. We wanted to also look at instantaneous DR increases and decreases between strips. The DR increase test used eight 3 cm wide strips with DRs increasing from (ΔMU/Δt)max to (ΔMU/Δt)max/32 in factors of 2 as the MLC bank moves from left to right. Unlike the DR increase test, there was some vertical pixilation in the images. Since we had two images for each measurement, any pixilation points  were deleted with the average between the two measurements including just the unpixilated portion. Deletion of vertical pixilation data was easily verified manually, since the profiles showed the single data spikes, and there were less than eight lines in each image.The effect of DR decrease was investigated as with DR increase, except the DRs decrease from ±1 mm in the cross‐plane direction  max/32 data.To avoid confusion in comparing dose rates with others, a nominal maximum DR is listed as 600MU/min, per Elekta guidance recommendation for entry into the TPS. Actual DRs for our LINAC, displayed during delivery, are given in Table ±0.5mm and 1 mm for the stationary cardinal angles and 180° arc, respectively max:(Δθ/Δt)max was delivered at (ΔMU/Δt)max/2:(Δθ/Δt)max/2. While the correct dose was delivered, we found that the strip would be delivered as planned with (Δθ/Δt)max of 5.86°/sec. The departure from 5.9°/sec, measured using a 90° arc, is likely due to the gantry speed ramp‐up time with the shorter arc used in this test (33°), which is controlled by the gantry inertia compensation distance parameter .Tests controlling DR and GS show accurate delivery within MV Table  with sta±3.5% with standard deviations on the same order of magnitude and occasionally larger. Dose to the 2D detector array was about 3.3 cGy, which is quite low. Cumulating the dose over repeated irradiations (up to 10) did not improve the results of the mean or standard deviation. We decided to repeat the test using the EPID which, while having less dose due to the larger source‐to‐detector distance, has increased resolution. A transverse profile of half an MLC width (0.5 cm) centered 0.5 cm superior to the central ray was used for the analysis, which minimized interleaf leakage considerations. The same method of normalizing the test measurement with the open beam profile per the initial test design was used, but measured using the EPID instead of the 2D array, for each DR. Some consideration was given over how many pixels to average over. The results were within ±2% tolerance with smoothing over 5 pixels (1.3 mm), but the standard deviation (estimated using the raw data) was quite large. Increasing the pixel averaging to 7, 9, or 11 improved the agreement between the different strips; there was much better agreement with increased pixels. The standard deviation of the raw data average over 7, 9, and 11 pixels was about the same, and was about half the value as that using 5 pixels. It was decided to smooth the data over a 7‐pixel moving average (1.8 mm at 100 cm SAD). Results showing the 7‐pixel average at the strip midpoint are shown in Table Initial tests controlling MLC leaf speed and DR show accurate delivery within (Δx/Δt)max, respectively. Underdosing was 0.4, 0.2, and 0.1% for the same (Δx/Δt), respectively.Normalized measurements of MLC reversals are given in Fig. >2% were seen for 6 MV DR increases between (ΔMU/Δt)max/2→(ΔMU/Δt)max, with a maximum of less than 3%. The extreme jump from (ΔMU/Δt)max/32→(ΔMU/Δt)max had doses >±3% over 9 mm of MLC leaf traversal (under‐ and overdose) max/32→(ΔMU/Δt)max having doses >±3% had a smaller transverse width of about 6 mm, due to a negligible overdose portion.Differences of se) Fig. . Resultsse) Fig. , except (ΔMU/Δt)max→(ΔMU/Δt)max/2 greater than 2% and 3% were seen over 6 mm and 2 mm transverse distance, respectively. The extreme case of (ΔMU/Δt)max→(ΔMU/Δt)max/32 was within ±2% max issues, the differences are not thought to be clinical significant. In hindsight, it is suggested in designing tests for each parameter to use 95% of the maximum value to avoid wasted time fixing nonclinically‐important findings such as a slightly lower GS (section C.1.1). This would be more efficient in allowing the same controlling files to be transported to additional LINACs. The tests should still include values beyond the maximum, as was done here to verify that the LINAC controller modifies values of MLC speed, DR, and GS needed to deliver the correct treatment.The differences between 6 and 10 MV in the physical parameters of MLC speed, backup‐jaw speed, and GS are measureable (Table (Δx/Δt)max/32:(ΔMU/Δt)max/16; (Δx/Δt)max/16:(ΔMU/Δt)max/8; (Δx/Δt)max/8:(ΔMU/Δt)max/4; (Δx/Δt)max/4:(ΔMU/Δt)max/2; (Δx/Δt)max/2:(ΔMU/Δt)max; (Δx/Δt)max:(ΔMU/Δt)max ; 1.2(Δx/Δt)max:1.2(ΔMU/Δt)max. Note this configuration would result in the last two strips having half the dose of the previous strips, which would complicate the analysis. This problem could be solved with the MLCs reversing direction after the first traversal to give a traversal in the opposite direction for just the last two strips with the same parameter values during the first traversal. Based on results from Fig. In testing MLC speed accuracy, significant time was added due to the low signal that made measuring the dose with the 2D diode array problematic which, while improved with the EPID measurements, still had signal issues as discussed in Section C.1.2. A different test design is suggested with the strips having twice the DR than that given in the Methods and Materials Section above, which will double the dose: The instant directional reversal of the MLCs was interesting Fig. ; and whi(ΔMU/Δt)max/2 or less, which gives DR change issues in the 1% region. You could argue that the excursion would be worse if we used a MLC speed of 2 cm/sec instead of 1 cm/sec, which would increase the transverse distance of the unacceptable doses reported here; but clinically, the MLCs move slower in areas that require dose that also requires higher dose rates, and move faster in areas not requiring dose with lower dose rates — which minimizes the significance of these results. The vendor is actively working on having a continuously‐variable DR, instead of the current binned‐DR parameter, which may minimize this issue in the near future. The tests presented here could still be used after this is provided, except for verification of maximum GS, MLC speed, and jaw speed which require the tester to see the drop in the dose rate by a factor of 2.The DR change measurements Figs.  and 10 hCommissioning VMAT for an Elekta linac using procedures given for RapidArc was carried out,Commissioning of VMAT should start with comprehensively testing the accurate operation of the linac, but must not be unduly burdensome for the busy clinical physicist. The testing suggested by Ling et al.The authors would like to acknowledge the kind assistance of Sergey Kulikov, MS, of Elekta, Ltd, for his demonstration on building iCOMCat files.


A host gene, normally an endogenous housekeeping gene  , or an irrelevant exogenous gene   has been widely used as an internal control to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. An endogenous internal control designed based on the 18S rRNA gene was used in the above-mentioned qPCR assay. This 18S rRNA internal control amplifies the target gene in multiple species including bovine, swine, ovine, caprine and cervine. Data was generated by the duplex qPCR assay on 138 enriched cattle lymph node samples without the internal control, and compared with data on the same samples tested by the triplex qPCR assay that has the 18S rRNA gene as internal control. Threshold cycle (Ct) data for the duplex and the triplex qPCR on the 138 samples were similar, and are presented in this brief report.A real-time PCR (qPCR) assay targeting on  Specifications tableValue of the data•Salmonella enterica detection.An internal control was used and showed value in monitoring nucleic acid extraction efficiencies in a real-time PCR assay for •Similar threshold cycle (Ct) data were generated with and without the use of the 18S rRNA gene as internal control.•Although an internal control is strongly recommended for real-time PCR assays in diagnostic settings, it may be optional to use for certain research projects like the one reported here.1Salmonella enterica detections is shown in invA and pagC genes; and 2) A triplex qPCR assay using invA, pagC, and 18S rRNA gene as an internal control. The Ct differences between the duplex and triplex qPCR assays are also presented in the last two columns in Real-time PCR (qPCR) test data on 138 culture-enriched cattle lymph node samples for 2Salmonella beads. One hundred microliters of PBS was added to the final immunomagnetic separation step. The bead suspension was then transferred into 3 mL RV broth and incubated at 42 °C for 18–20 h. One hundred microliters of enriched homogenate was streaked onto HE agar plates and incubated at 37 °C for 24 h. Six dark-colored colonies with morphology consistent with Salmonella were re-streaked onto BAPs and incubated at 37 °C for 18–20 h. The resulting cultures was used for DNA extraction by boiling 1 ml of culture for 10 min and centrifuging at 9300 g for 5 min; the supernatant was used as template for the qPCR reactions with the duplex qPCR assay using both invA and pagC genes as molecular targets. Endogenous housekeeping genes A total of 647 subiliac lymph nodes were collected from cattle procured from Texas, Oklahoma, Kansas, and South Dakota


Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2’-O-methyl (2’-OMe) modification at their 3′ terminal nucleotide. This inhibits 3′ adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina’s classical TruSeq protocol regarding the detection of normal and 2’ OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance.Among the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2’ OMe RNAs both in the TruSeq as well as in the NEXTflex protocol.While it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2’ OMe RNAs.The online version of this article (10.1186/s12864-018-4491-6) contains supplementary material, which is available to authorized users. Small RNAs (sRNAs) are known to play an important regulatory role in a wide range of organisms in many biological processes including embryo development, cell differentiation, growth/proliferation and apoptosis/cell death . EukaryoNext-generation sequencing (NGS) is a powerful tool for the analysis of sRNAs. It has several advantages over microarray techniques or quantitative PCR (qPCR). It allows for the discovery of novel sRNAs, has a better signal to noise ratio than microarrays and does not suffer of saturation effects. In addition, it allows for the detection of single base differences and has a higher throughput than qPCR or Northern blotting. However, NGS approaches also have some disadvantages, such as the cost per sequencing run and the extensive processing steps required to convert a sample into a library for sequencing. In a typical sRNA library preparation process, adapters are ligated to the RNAs followed by reverse transcription and PCR amplification. All these steps are potential sources of bias , 4. ConsSeveral studies demonstrated that adapter ligation steps may cause serious bias, due to RNA sequence/structure effects resulting in the preferential ligation of certain sRNAs with a given adapter sequence, while others are disfavoured –11. We wInstead of modifying the adapters, some other studies sought to suppress bias through the optimisation of reaction conditions. Several laboratories tested a thermostable DNA/RNA ligase, ‘MthRnl’ from New England Biolabs that works at 65 °C, a temperature at which short RNAs should be largely unstructured. Even though one study reported bias reduction by this enzyme , two othHowever, all these results were obtained with unmodified RNAs and do not necessarily extrapolate to 2’ OMe RNAs. One paper reported that the addition of 25% PEG restored ligation efficiency of a 2’-OMe modified sRNA to the level of the same RNA without modification [Another concern for sRNA library preparation is the formation of side products such as adapter dimers. Currently available library reparation kits either use strategies to eliminate excess 3′ adapter before 5′ adapter ligation, including purification steps or the use of complementary oligonucleotides that inactivate the 3′ adapter. Several alternative approaches have been developed. For example, Shore et al.  developeSome protocols use polyadenylation instead of ligation for 3′ adapter addition. Multiple A residues are added to the 3′ end using poly(A) polymerase (PAP) . It remaIn this study, we systematically compared the classical TruSeq kit from Illumina with four commercially available ‘low bias’ kits, the NEBNext kit from NEB, the NEXTflex V2 kit from BIOO Scientific, the SMARTer kit from Clontech and the CATS kit from Diagenode. We tested the performance of these kits with regard to three parameters: (1) bias among sequences, (2) bias against 2’ OMe RNAs, and (3) the formation of side products. The ideal protocol should have low overall bias and generate few side products. This would allow to faithfully reproduce true sRNA expression profiles and to capture (weakly expressed) sRNAs that might otherwise escape detection. We tested the kits with and without several modifications, and using human miRNAs as representatives of non-modified sRNAs and plant miRNAs representing 2’ OMe-modified sRNAs. Our results identify protocols that work best for these different types of sRNA. Our modifications lead to better detection of 2’ OMe RNAs than the standard protocols.To test the performance of the various kits, we designed a pool of six synthetic sRNAs, supplemented with random 21 nt RNAs to create a complex mixture. As ligation efficiency can be influenced by RNA structure effects, the six synthetic sRNAs (RNAs1–6) were designed with various predicted secondary structures , Fig. 1.We started by comparing the levels of bias introduced by the various kits following their respective standard protocols. We determined the relative proportion of reads corresponding to each of the RNAs(OMe)1–6 considering the total numbers of raw reads before adapter trimming to take into account also potential losses of sequences due to the formation of adapter dimers , PEG was added to the ligation reactions to a final concentration of 20% in combination with the Illumina adapters (TS3), or in combination of HD adapters (TS4). With the NEXTflex protocol, the reverse experiment was performed; the HD adapters were replaced by Illumina adapters (Nf3) or PEG was left out of the reaction mixtures while keeping the HD adapters (Nf4). Note here that the standard NEXTflex protocol uses HD adapters in combination with PEG.For both kits, HD adapters present less sequence bias than Illumina adapters, as previously reported –11  that did not correspond to any of the RNA(OMe1–6) or to the 21 nt random RNAs  than with the NEXTflex kit (Nf2). Our results further indicate that small changes in protocols, even when keeping the same adapters, can have profound effects on expression profiles.Fuchs and colleagues developed a variant of HD adapters, called ‘MidRand’, in which the randomised regions are in the middle of the adapters rather than at the extremities . In addiThe strong bias observed with the classical TruSeq protocol raises questions of the capacity to detect expression changes of a given sRNA in different conditions. It was previously reported that despite strong sequence bias 10–1000 fold changes were well detected , and a sB. napus sRNA preparations from either stems and leaves or from flower buds, and miRNA expression changes were measured. As shown in Additional file To further confirm these observations, we selected a few protocols  to prepare libraries using To summarise the above sections, our analysis with the synthetic RNAs revealed that the SMARTer and CATS kits had less bias than the standard TruSeq and NEXTflex protocols. However, upon modification, bias levels with the TruSeq kit strongly decreased and dropped below those obtained with the SMARTer and CATS kits. MRL adapters introduce less bias than HD adapters with both the TruSeq and NEXTflex kits. Unfortunately, low bias conditions also favoured the formation of side products. There was a striking reverse correlation between bias levels and side product formation, and the performance of a protocol will thus depend on a balance between these two. For 2’ OMe RNAs there is, next to sequence bias, also 2’ OMe bias to be taken into account in this balance. As a result, protocols that perform best for unmodified RNAs may not be the same as those that yield the best results for 2’ OMe RNAs.Arabidopsis miRNAs, that are known to contain a 2’OMe modification. We assessed the protocols for three different aspects: (1) the percentage of sequencing reads corresponding to miRNAs, (2) the formation of side products, and (3) the number of different (i.e. unique) miRNAs detected. The libraries were sequenced using Illumina technology and the reads were trimmed for adapter sequences and mapped to human or Arabidopsis miRNA databases  and short side products (S). With all protocols, the percentage of mapped reads remained modest, which is consistent with literature data [Arabidopsis material than in the human RNA sample.Strikingly, for ure data , 24. ThiWe next asked which protocol would allow to detect the widest possible range of different miRNAs. As this is likely to depend on the sequencing depth, we considered the same number of reads (one million) for all libraries sequenced. The numbers of miRNAs mentioned are mean values from at least two independent experiments. With TS1, 690 known human miRNAs were identified, which represents ~ 27% of the total number in miRBase (2588 human miRNAs) Fig. . The lacArabidopsis, the TS1 protocol detected 142 known miRNAs, which represents ~ 33% of the total number of miRNAs registered in miRBase (427). As for the human miRNAs, protocols TS2 and TS4 performed less well, but in contrast TS3 performed better than TS1, with 201 miRNAs detected. Another difference was that for Arabidopsis the best results were obtained with protocol TS6 (217 miRNAs identified). Both in the TruSeq as well as in the NEXTflex kit, MRL adapter performed better than HD adapters . The SMARTer protocol detected 161 miRNAs, slightly better than TS1. To validate these observations, the same experiment was done with Brassica napus (oilseed rape) RNA . Very similar results were obtained as for Arabidopsis, underscoring the reproducibility of the data  human miRNAs the standard NEXTflex protocol performed best, whereas modification of the TruSeq protocol led to only a modest improvement. For the (2’ OMe modified) plant miRNAs, modification of the TruSeq protocol led to a more marked improvement of detection and the best results were obtained with MRL adapters in both kits.Arabidopsis libraries following protocols TS1, − 5, − 7, Nf1, − 6, and S, and generated 20 million reads for each library. We determined the number of known miRNAs identified at increasing read numbers ranging from 100 thousand to 20 million reads  causing some sRNAs to be strongly detected while others are underrepresented. It is therefore possible that each protocol has its own specific subpopulation of miRNAs that are well represented. To address this question, we compared the miRNA expression profiles for the different protocols. Additional file We asked if a combination of different protocols might help to further increase the number of different miRNAs detected. To test this possibility, we compared the number of different human miRNAs detected with 1 million of reads for protocol Nf1  miRNAs the standard NEXTflex protocol performed best, and the modifications we introduced had little effect. In contrast, for plant miRNAs (2’ OMe modified) these modifications substantially improved detection, and the best results were obtained with PEG and MRL adapters in both the TruSeq and the NEXTflex kit. Our results thus yield improved conditions for the detection of 2’ OMe RNAs and in addition indicate that different protocols work best for different types of sRNA.It should be mentioned here that we have not exhaustively tested all sRNA library preparation kits available on the market. However, at least to our knowledge, none of these other kits use randomised adapters and are therefore likely to have more bias than the NEXTflex, SMARTer and CATS kits. It should be kept in mind that despite the strong bias, the standard TruSeq protocol can quantitatively detect even small expression changes of both normal and 2’ OMe RNAs, provided that these are well detected.http://unafold.rna.albany.edu/?q=mfold) [All oligonucleotides used in this study were purchased from Sigma-Aldrich or BIOO Scientific (HPLC purified). A series of six synthetic small RNAs, RNA 1–6, was purchased from Sigma-Aldrich. Of each RNA a 2’-OMe variant (RNA-OMe 1–6) carrying a single nucleotide (nt) substitution (to distinguish the 2’-OMe variant from the unmodified RNA) was purchased. To predict the secondary structures of RNAs1–6 alone, linked to the 3’ Illumina adapter, or linked to both the 3′ and 5’ Illumina adapters , we used the Mfold web server (q=mfold) . The strq=mfold) .A mix of random 21 nt RNA oligonucleotides was purchased from BIOO Scientific. All of these RNAs carried a 5′ monophosphate. RNAs 1–6 and their 2’ OMe variants were added to the mix of random RNAs to a final concentration of 0.1 μM each .In some of the library preparation protocols (see below), the adapters included in the kits were replaced by custom adapters; we used custom HD adapters and MRL adapters. The 3′ adapters were pre-adenylated following a protocol described by Chen et al. ; 1 nmol Arabidopsis thaliana (ecotype Col-0) and Brassica napus  were grown under controlled conditions (18 °C during 8 h at night and 21 °C during 16 h during the day) in the same climate chamber. Samples were collected all at once for each species. Total RNAs were extracted from young leaves and stems or flower buds using the TRIzol® reagent following the manufacturer’s protocol (Invitrogen), with addition of 0.2 μL/mL beta-mercapto-ethanol to TRIzol® extemporarily. Human RNA (HeLa) was from the Total RNA-seq kit from ThermoFisher.Individual plants for the two plants species Arabidopsis, oilseed rape or human (HeLa) was migrated on 15% polyacrylamide gels containing 8% urea alongside with molecular size markers. RNA was visualized with SYBR Gold (Life Technologies) and 15–30 nt RNAs were excised from the gel. The gel pieces were crushed by centrifugation using 0,5 mL Eppendorf tubes with tiny holes at the bottom. These tubes were put in 1,5 mL Eppendorf tubes followed by 2 min centrifugation at 13 krpm. Subsequently 300 μL 0,3 M NaCl was added to the crushed gels and RNA was eluted by rotation overnight at 4 °C. RNA was ethanol precipitated and resuspended in 15 μL water. We used 1 μL of synthetic RNA mix  or 1 μL of gel-purified biological small RNA  for library preparation.All protocols used in this study were based on the Illumina TruSeq Small RNA Sample Preparation kit, the BIOO Scientific NEXTflex Small RNA Sample Preparation kit V2, the New England Biolabs NEBNext kit, the SMARTer smRNA-seq kit from Clontech, or the CATS kit from Diagenode. As starting material either the above-described mix of synthetic RNAs was used (10 pmol RNA per reaction) or biological small RNA samples. Biological small RNA samples were prepared as follows: 10 μg of total RNA from TS1. TruSeq – standard conditions. Library preparation was done using the Illumina TruSeq Small RNA library preparation kit following the manufacturer’s instructions, except that for 3′ adapter ligation 1 μL of synthetic or biological small RNA was mixed with 1 μL RA3 adapter followed by 2 min’ denaturation at 70 °C. After this denaturation step the samples were put on ice and 4 μL of water, 2 μL of ligation buffer, 1 μL of RNA ligase 2 truncated (New England Biolabs) and 1 μL of RNaseOUT was added.TS2. TruSeq + HD adapters. The same procedure as for TS1 except that the Illumina adapters RA3 and RA5 were replaced by HD adapters.TS3. TruSeq + PEG. The same procedure as for TS1 except that after the denaturation step for 3′ adapter ligation 4 μL of PEG8000 was added instead of water and ligation was done at 16 °C overnight instead of 1 h at 28 °C.TS4. TruSeq + PEG + HD adapters. The same procedure as for TS3 except that the Illumina adapters RA3 and RA5 were replaced by HD adapters.TS5. TruSeq + PEG + HD adapters + purification. The same procedure as for TS4 except that, instead of adding STOP solution, a purification step was performed after 3′ adapter ligation to get rid of excess 3′ adapter. Two rounds of purification were done using AMPure beads (Beckman Coulter) as described in the protocol of the BIOO Scientific kit, except that the ligation products were eluted in 9 μL H2O instead of 11 μL. Subsequently 2 μL of Illumina ligation buffer was added for 5′ adapter ligation.TS6. TruSeq + MRL adapters. The same procedure as for protocol TS2 except that MRL adapters were used instead of HD adapters.TS7. TruSeq + PEG + MRL adapters + purification. The same procedure as for protocol TS5 except that MidRand adapters were used instead of HD adapters.Nf1. NEXTflex protocol. Library preparation was done using the BIOO Scientific NEXTflex Small RNA-Seq kit V2 following the manufacturer’s instructions with 3′ adapter ligation for 2 h at 22 °C.Nf2. NEXTflex protocol: as for protocol Nf1 but with 3′ adapter ligation overnight at 16 °C.Nf3. NEXTflex protocol w/o HD adapters: the adapters from the NEXTflex kit were replaced by the Illumina adapters.Nf4. NEXTflex protocol w/o PEG: PEG was replaced by water.Nf5. NEXTflex protocol + MRL adapters w/o PEG: the adapters from the BIOO kit were replaced by MRL adapters and PEG was replaced by water.Nf6. NEXTflex protocol + MRL adapters: the adapters from the BIOO kit were replaced by MRL adapters.NN. NEBNext protocol: Library preparation was done following the manufacturer’s instructions with 3′ adapter ligation for 1 h at 25 °C.S. SMARTer protocol: Library preparation was done using the standard protocol provided with the kit. For the libraries from the synthetic RNA mix ATP was added to the polyadenylation reactions and 7 cycles of PCR were done. For the libraries from the biological samples the polyadenylation reactions were done without supplemented ATP and 13 cycles of PCR were done.C. CATS protocol: Library preparation was done following the manufacturer’s instructions.http://www.mirbase.org/ftp.shtml ; we only used the mature sequences of Arabidopsis thaliana, Homo sapiens and Brassica napus. For piRNA detection, the reads of the human libraries were mapped to piRBase . The sampling of the reads was done using seqtk, version 1.0-r31 . Only the reads with a length between 19 and 24 nt were kept using python scripts. Mapping to the databases was performed with Bowtie2, version 2.1.0, [All sequencing was done using Illumina platforms, either the MiSeq or the NextSeq500 instrument. For each protocol, several independent libraries were prepared; the mean values are represented in the Figures. Cutadapt  version n 2.1.0,  allowingArabidopsis RNA, the obtained sequences were trimmed for adapter sequences and were mapped to various databases. For human, the sequences were mapped to the database of small human non-coding RNAs  [http://www.regulatoryrna.org/database/piRNA/download/archive/v1.0/fasta/) for piRNA sequences. The Arabidopsis sequences were mapped to databases for various non-coding RNAs;ftp://ftp.ensemblgenomes.org/pub/release-24/plants/fasta/arabidopsis_thaliana/ncrna/Arabidopsis_thaliana.TAIR10.24.ncrna.fa.gz), tRNAs  and miRNAs (miRbase). All of these databases were downloaded on October 6, 2017.For the annotation of the short side products (10–18 nt inserts) obtained with the SMARTer libraries from human or 5, 2017) , mirBaseTo generate the heat map shown in Additional file Additional file 1: Figure S1.total RNA) or RNA-OMe1–6 . Shown are the mean values of at least two independent experiments. Error bars represent standard deviations. Note that in the absence of bias or loss of sequences, for each individual RNA the percentage of the total number of reads should be 1%, and for the sum of the unmodified or the 2’ OMe RNA this percentage should be 6%. For each RNA the ratio of the read numbers for the unmodified- and the 2’ OMe variant is indicated below the histograms . (PDF 1.01 mb)Histograms representing the percentage of the total numbers of raw reads (before trimming) corresponding to RNA(OMe)1–6 with the TruSeq protocols TS1–7, the NEXTflex protocols Nf1–6, the NEBNext protocol (NN), the SMARTer protocol (S) and the CATS protocol. Blue bars represent the numbers of reads corresponding to each individual RNA, red bars represent the numbers of reads corresponding to RNA1–6  corresponding to RNAs 1–6 with the various protocols. In the ideal situation, 16,7% of the reads (indicated by a red line) should correspond to each RNA, without significant variability among the different RNAs. The data for each individual replica of the various protocols are shown. Horizontal black bars indicate the median RNA (MR). (PDF 1.01 mb)Additional file 3: Figure S3.Polyacrylamide gel analysis of ligation products of RNA1, RNA1-OMe, RNA4 and RNA6. Samples were taken after 3′ and subsequent 5′ adapter ligation followed by electrophoretic separation on a 10% denaturing polyacrylamide gel. Mixtures of the synthetic RNAs and 3′ adapter without ligation (−) were migrated along with the ligation products. Unligated 3′ adapter and synthetic RNAs, which almost co-migrated in the gels, are indicated by an accolade. Asterisks indicate from bottom to top: unligated 5′ adapter, RNA ligated with 3′ adapter, RNA ligated with 5′ adapter, and RNA ligated with both adapters. Note that RNA ligated with 3′ adapter migrates faster than RNA ligated with 5′ adapter because the 3′ adapter (21 nt) is smaller than the 5′ adapter (26 nt). (PDF 1.01 mb)Additional file 4: Figure S4.Histograms representing the percentage of the total numbers of raw reads corresponding to RNA(OMe)1–6 with (A) the TruSeq protocol (TS1), (B) the NEXTflex protocol (Nf2), and (C) the SMARTer protocol (S). Green bars represent the results obtained with the standard numbers of PCR cycles , blue bars represent 50 additional cycles of PCR, and red bars represent the standard number of PCR cycles but using AMV reverse transcriptase instead of Superscript II for cDNA synthesis. (PDF 1.01 mb)Additional file 5: Figure S5.2) were determined for the fold changes obtained with each protocol for the unmodified RNAs  and for the 2’OMe RNAs  separately, and for the collection of the unmodified and 2’ OMe RNA together (R2tot). The fold changes obtained with the various protocols were compared with the theoretical values and the different protocols were compared to each other. (PDF 1.01 mb)Assessment of quantitative detection of synthetic RNAs with protocols TS1, TS5, Nf2, or S. Libraries were prepared from a synthetic RNA mixture in which RNA(OMe)1–6 were each at 1% final concentration, supplemented with random 21 nt RNAs (mix A). Alternatively, an RNA mixture was used in which the concentrations of RNA(OMe)1–6 were changed (mix B); see table Additional file 6: Figure S6.B. napus small RNA preparation originating either from floral buds or from stems and leaves and miRNA expression changes were measured. The detected fold changes with the different protocols were compared to each other and the coefficients of determination (R2) were calculated. (PDF 1.01 mb)Quantitative detection of oilseed rape miRNAs using protocols TS7, Nf2, and Nf6. Libraries were preparing from Additional file 7: Figure S7.Arabidopsis libraries. After adapter trimming the human sequences were mapped to the database of small human non-coding RNAs (DASHR), mirBase , and the regulatory RNA database for piRNA sequences. The Arabidopsis sequences were mapped to databases for various non-coding RNAs (Ensemblgenomes), tRNAs (Genomic tRNA database) and miRNAs. See Annotation data for the short side products (10–18 nt inserts) with human and Additional file 8: Figure S8.Arabidopsis miRNAs identified with different thresholds of coverage. We determined the numbers of known miRNAs identified with the various protocols for human or Arabidopsis with a minimum coverage of 1 read per miRNA as shown in Fig. Arabidopsis) independent experiments with standard deviations represented by error bars. (PDF 1.01 mb)Numbers of known human or Additional file 9: Figure S9.Numbers of known human piRNAs identified. We determined the numbers of known piRNAs identified with the various protocols. For each protocol, one million of reads were trimmed and the 19–24 nt inserts were used for mapping to human piRNAs in piRBase Additional file 11: Figure S10.Heat map representation of miRNA expression profiles obtained with protocols TS1, TS3, TS5, TS7, Nf1, Nf2, Nf6, and S. We determined the proportion of reads mapping to each miRNA as a percentage of the total number of mapped reads. These proportions are represented by a colour spectrum from very light red (weak expression) to dark red (strong expression). Shown here are the results for the 50 most highly expressed miRNAs, and the sequences and names of the miRNAs are indicated on the right. Data for all detected miRNAs are shown in Additional file Additional file 12: Table S2.Oligonucleotides used in this study. (XLSX 9 kb)


In particular, a circular dichroism of ~40° and a maximum polarization rotation of ~200°/λ are observed around 0.7 THz. Furthermore, our study also reveals that the chiroptical response from the proposed metamaterials is strongly dependent on the phase transition of VO2, leading to actively controllable polarization states of the transmitted terahertz waves. This work paves the way for the development of terahertz metadevices capable of enabling active polarization manipulation.Compared with natural materials, chiral metamaterials have been demonstrated with orders of magnitude stronger chiroptical response, which provides the basis for applications such as ultracompact polarization components and plasmonic-enhanced biosensing. Terahertz chiral metamaterials that allow dynamic polarization control of terahertz waves are of great practical interest, but remain extremely rare. Here, we show that hybrid metamaterials integrated with vanadium dioxide (VO Moreover, chirality based manipulation of the polarization state of light has been exploited in various optical applications such as Faraday isolators2, which depend on the Faraday effect induced by an external magnetic field. However, due to the extremely weak chirality of naturally occurring media, bulk chiral materials are generally required to obtain a detectable chiroptical response.Chiroptical responses, such as circular dichroism (CD) and optical activity, observed in natural materials, in general reflect the corresponding structural symmetry inherent at the microscopic level. Accordingly, circular polarization has been utilized to generate, transmit and measure electromagnetic signals that carry important information during the light-matter interaction process. For example, CD spectrum spectroscopy, which is based on the difference in the absorption of circularly polarized light of opposite handedness, has been widely utilized in biosensing for the extraction of structural information of large biological moleculesi.e., the meta-atoms5. In particular, metamaterials comprised by meta-atoms lacking mirror symmetry have been implemented to realize a giant chiral response23. Such structures offer extensive potential for applications based on the far-field and near-field chiroptical effect, such as circular polarizers19, polarization rotators22 and plasmonic-enhanced biosensors24. Analogous to their counterparts for the manipulation of linearly polarized waves, conventional chiral metamaterials derive their resonant properties from the geometric arrangement of their meta-atoms and their constituent material composition. One consequence of this, however, is the fact that their chiroptical responses are generally fixed. To further functionalize metamaterials, hybridizations of active media  with conventional metallic resonating components have been implemented to enable reconfigurability via external stimuli (see the recent review papers26). Recently, germanium antimony telluride (Ge2Sb2Te5 or “GST”), a type of phase change material, has been harnessed to facilitate thermally tunable chirality for chiral plasmonic nanostructures operating in the mid-infrared regime28.Metamaterials have been shown to offer unprecedented flexibility in manipulating electromagnetic waves through design and tailoring of their subunit building blocks, 47, have proven to be highly effective in manipulating THz radiation in ways that are far superior to other natural media and therefore offer a tremendous potential for the creation of new and transformative THz devices. Among them, chiral metamaterials that provide the ability to dynamically control the polarization state of THz waves are of great importance for THz metadevices with sophisticated functionalities, but such examples remain rare. Photocarrier excitation enabled dynamic control of chirality has been reported in semiconductor based active THz metamaterials. In particular, Kanda and coauthors have demonstrated optical pumping induced THz optical activity in a semiconductor substrate which was masked by a two-dimensional array of chiral meta-atoms33. Zhang and coauthors have reported the handedness switching in three-dimensional THz chiral metamaterials in response to an external optical stimulus36. Zhou, et al., have verified that a variety of optically tunable chiral responses, including a chirality induced negative index of refraction, can be produced by a gold/silicon bilayer conjugate chiral microstrucuture38. Moreover, Lv, et al., have demonstrated a multiband background-free THz switch in a silicon filled bilayered chiral metamaterial45. Nevertheless, THz metamaterials capable of producing strong chiroptical responses that can be dynamically tuned over a large scale are highly sought after.Terahertz (THz) metamaterials, especially those endowed with tunability2), which undergoes an insulator-to-metal transition (IMT) near room temperature (TIMT ~67 °C), has been intensively studied due to its potential applications in both electronic and optical devices. Recently, VO2 has been introduced into metamaterial systems to enable response modulation of various nano-50 and micro-structured systems47 to external stimuli, such as temperature and electric current. Lv and coauthors have theoretically reported the observation of thermally tunable linear polarization conversion in a THz metamaterial composed of VO2-loaded twisted E-shaped metallic resonators47. We emphasize that besides an approximately three orders of magnitude increase in the THz conductivity (σ1) during IMT, the reported data40 indicates that σ1 of VO2 in its metallic state can be as high as 4 × 103 Ω−1cm−1, which is roughly one order of magnitude lower than that of gold. Consequently, VO2 is able to support a strong THz resonance that can be exploited to achieve highly tunable THz devices. Recently, the temperature-tuned transmission of THz waves has been observed in metamaterials consisting of VO2 cut-wires51. We have also shown that the integration of VO2 structures with conventional metallic resonating components can enable a class of highly tunable THz metamaterials52.Experiencing atomic level deformation, phase change materials (PCMs) are capable of offering drastic variations in material properties over a broad spectral range during the phase transition. As a representative of PCMs, vanadium dioxide  in addition to optical resonance, which provides the basis for realizing ‘self-sufficient’ metadevices54. However, a close inspection of the reported chiral metamaterials reveals that most designs cannot support the additional functionalities because they typically consist of geometrically isolated units20. With a topologically continuous layered structure, twisted fishnet metamaterials are considered as good candidates for multifunctional chiral metadevices. By measuring the complex scattering parameters of a stack of twisted elliptical hole arrays, Wang, et al., have demonstrated large optical activity and circular dichroism at THz frequencies55. By implementing a scaled-down structure, Kang, et al., have demonstrated a photonic metamaterial with nonlinear chiral-selective electro-optic functionalities54. In addition, Rodrigues, et al., have reported intensity-dependent optical rotation in a similar twisted fishnet nanostructure22.Since the two enantiomers of chiral objects exhibit identical scalar physical properties, remotely sensing the chiroptical response of an object provides a unique approach to enantioselectivity due to the fact that circularly polarized light (CPL) itself is chiral. Given the generally weak interaction between CPL and natural media, artificially structured materials have a long history of development with the goal of enhancing light-matter interaction and achieving stronger chiroptical effects. Owing to their resonant properties, the recent introduction of metamaterials offers unprecedented flexibility for realizing enormously strong chiral responses. Following the general design strategy, 2 in its metallic state, i.e., σ1 = 3×103 Ω−1cm−1 (see Methods for details)40. Figure 2/gold twisted fishnet metamaterial structure, along with the unit cell schematics of the two enantiomers which are mirror images of each other. A 3 μm-thick VO2 film and a 500 nm-thick gold film are perforated by identical elliptical holes (a = 60 and b = 40 μm) with angular offsets of α = 22.5° and β = 45° and separated by a dielectric spacer (ts = 42 μm). It should be noted that in order to support a strong resonance in its metallic state, the VO2 film should not be thinner than the corresponding skin depth, which is ~1 μm around 1.0 THz. The unit cell is arranged in a two-dimensional square lattice with a lattice constant of 130 μm. The 2D projection plots of the two enantiomers clearly indicate the chiral symmetry of the meta-atoms. The transmission spectra of left- and right-handed circularly polarized (LCP and RCP) waves are illustrated in Fig. i.e., RCP in Fig. i.e., 0.65, 0.72 and 0.82 THz, where the relatively high transmission amplitude of the LPC and RCP waves are balanced. We note that the polarization rotation capability at multiple frequencies can be beneficial for multi-channel THz polarization components. To better illustrate the observed chiroptical phenomena, the corresponding circular dichroism (CD) and optical rotatory dispersion (ORD) are depicted in Fig. tan−1[(tR − tL)/(tR + tL)] and ORD = 0.5[arg(tR) − arg(tL)], where tR and tL denote the complex transmission coefficients of RCP and LCP waves, while arg represents the phase angle. It can be seen that a maximum value of CD as large as ~44° (~−44°) is achieved in enantiomer A (B) around the excited resonance of the LCP (RCP) wave. Moreover, for the enantiomer A (B), ORD reaches ~30°, −27° and 6°  at the three critical frequencies mentioned above. Considering the subwavelength thickness (~λ/9) of the metamaterial, a maximum polarization rotation of ~240°/λ around 0.7 THz is expected.It has been shown that the giant chiral response from the twisted fishnet structures arises from the strong electromagnetic coupling between the two conductive layers under circular polarized illumination. Consequently, we start our design by adopting the THz conductivity of VO2 and Au layers for enantiomer A are shown in Fig. e.g. LCP) wave, the explicit field distribution at different resonances reflects the dispersion characteristic of the chiral metamaterial. Furthermore, referring to the CD spectra shown in Fig. The chiral-selective transmission spectra shown in Fig. 2 experiencing a dramatic change in conductivity during the IMT. We note that the response of patterned VO2 film has been previously studied in the optical regime, for instance in ref.48. However, the corresponding structured VO2 in its metallic phase cannot support a strong resonance due to the rather high loss exhibited at optical frequencies. Consequently, the observed resonance tuning of the metallic structures can be regarded as a result of the variable index provided by the VO2 when in a different material phase. In our work, the resonance arising from the coupling between the structured VO2 film and the perforated gold layer gives rise to the strong chiral response. Figure 2 conductivity values. First of all, when the VO2 is nearly in its insulator state (σ1 = 20 Ω−1cm−1), the metamaterial manifests weak chirality, i.e., almost no difference in the transmission spectra of LCP and RCP waves , the strong optical activity observed in chiral metamaterials does not require the application of an external magnetic field and will potentially find applications in polarization manipulating components, such as ultracompact polarization rotators. Therefore, hereinafter we focus on the actively controllable optical rotation properties of the proposed metamaterial. It should be noted that Wang and coauthors55 have reported large THz optical activity of a similar gold based twisted fishnet structure, while, however, the corresponding transmission was rather low. As the results shown in Figs Optical activity  represents the ability of certain materials to rotate the polarization plane of a linearly polarized wave. Different from the Faraday effect  for enantiomer A and enantiomer B in our study is chosen to be 45° and 135°, respectively, as depicted in Fig. θt and θin, where θt denotes the angle between the long axis of the polarization ellipse of the transmitted wave and the x axis. Figure 2 in its metallic state. To obtain a complete picture of the polarization state of the transmitted waves, the corresponding ellipticity, defined as i.e., 0° (45°), is obtained when the transmitted wave is circularly (linearly) polarized. It can be seen that the ellipticity reaches 45° at the frequencies of 0.62, 0.72 and 0.81 THz, indicating the linear polarization of the transmitted wave at these frequencies. As a result, in the following discussion we focus on these critical frequencies.It has been experimentally verified that a linearly polarized wave with its polarization plane parallel to the direction where the two perforated ellipses intersect most closely experiences the strongest optical activity2 conductivity are presented on semi-log plots in Fig. 1 increases the rotation angle of enantiomer B precisely exhibits opposite behavior compared to that of enantiomer A, once again confirming the enantiomeric characteristic of the metamaterials. Interestingly, for enantiomer A with VO2 in its insulator state (σ1 = 10 Ω−1cm−1), a polarization rotation angle of ~−10° and ~10° is observed at 0.62 and 0.81 THz, respectively. It should be noted that, as discussed above, this observation, which deviates from the results shown in Fig. 1, this characteristic may in fact result in a corresponding polarization rotation modulation as large as ~30° (from ~−10° to ~20°). Another interesting observation is that the rotation angle at 0.72 THz increases from ~0° to ~7° when σ1 increases in the range from 10 Ω−1cm−1 to 100 Ω−1cm−1, while it decreases dramatically to ~−20° when σ1 approaches 3×103 Ω−1cm−1. In other words, an optical activity switching can be achieved by controlling the phase transition of VO2. On the other hand, the change in σ1 may also tune the ellipticity of the transmitted waves, as displayed in Fig. The polarization rotation angle and ellipticity as a function of the VO56 and optical pumping57, electric current and field-effect-induced phase transition in VO2 have also been reported59. Despite controversy over their microscopic origins, field-effect based transition is considered to have the potential to achieve VO2 property modulation at the subpicosecond level58. On the other hand, electrically controlled active metamaterials are of crucial importance due to their potential for a wide range of practical applications. In this respect, VO2 integrated hybrid metamaterials that exhibit electrically triggered active responses have attracted great attention. For instance, by integrating split ring resonators (SRRs) and VO2 thin films, Driscoll, et al., have demonstrated electrically controlled memory effects at THz frequencies31. Goldflam, et al., have reported electrical signal controlled spatial gradient tuning in a THz metamaterial consisting of an array of SRRs located on a VO2 layer34. Liu and coauthors demonstrated a VO2 integrated photonic metamaterial for electrically triggered multifunctional control50. To this end, as we discussed above, the topologically continuous structure of the proposed metamaterial provides the possibility of achieving response modulations through electrically induced phase transition in VO2. Furthermore, it is worth noting that, as a classical transition metal oxide, VO2 exhibits hysteresis behavior during its IMT. Therefore, the chiroptical responses produced by the proposed chiral metamaterials are expected to exhibit hysteretic characteristics, which means that, for example, the corresponding optical activity observed would be system history dependent. Taking into account this hysteretic behavior, the proposed metamaterial is expected to manifest a certain memory effect and, compared with the memory process observed in other metamaterial systems50, its chiral-selective properties may provide more degrees of freedom for designing metadevices with electro-optic information processing and storage capabilities. Moreover, given the Drude-type dispersion model of metallic-phase VO2 in the THz regime, the actively controllable response of the proposed hybrid metamaterial can be geometrically scaled to other frequencies.Besides direct thermal control2/metal twisted fishnet structures can enable dynamically tunable chiroptical responses at terahertz frequencies. Simulations show that, with VO2 in its metallic phase, a circular dichroism of ~44° and a maximum polarization rotation of ~240°/λ can be achieved around 0.7 THz. More importantly, we found that the chiroptical responses, such as circular dichroism and optical activity from the hybrid metamaterials are closely dependent on the phase transition related THz conductivity variation of VO2. Considering the hysteresis and ultrafast dynamics characteristics observed during the phase transition of VO2, we envision that the proposed hybrid chiral metamaterials may potentially enable chiroptical platforms for complicated manipulation of THz radiation.To summarize, we have demonstrated that hybrid metamaterials based on VO2 in the THz region can be described by the following Drude model: ε∞ is the permittivity at high frequency, ωP(σ1) is the conductivity (the imaginary part of the complex conductivity σ2 is assumed to be 0) dependent plasmon frequency and γ is the collision frequency60. A close examination reveals that both σ1 are proportional to the free carrier density and, as a result, the plasmon frequency at 40, we have determined that when σ1 = 3 × 103 Ω−1cm−1, it follows that ωp = 1.40 × 1015 rad/s, while γ = 5.75 × 1013 rad/s is assumed to be independent of σ1. We note that these Drude model parameters are in good agreement with those reported by other groups60.It has been experimentally verified that the permittivity of VOi.e., tRR, tLR, tRL and tLL can be expressed as a function of four linear transmission coefficients of the chiral medium, i.e., txx, tyx, txy and tyy, such that i.e., Full-wave simulations were implemented using the commercial finite integration package CST Microwave Studio. A unit cell of the investigated structure was simulated by employing periodic boundary conditions. The four transmission coefficients of circularly polarized waves, The data that support the findings of this study are available from the corresponding author on request.


Determination of the mechanical energy storage performance of the aluminum fumarate metal–organic framework A520. The aluminum fumarate MOF A520 or MIL-53–FA is revealed to be a promising material for mechanical energy-related applications with performances in terms of work and heat energies which surpass those of any porous solids reported so far. Complementary experimental and computational tools are deployed to finely characterize and understand the pressure-induced structural transition at the origin of these unprecedented levels of performance. This solid, denoted as MIL-53(Al)–FA, was revealed to be isoreticular of the well documented highly flexible MIL-53(Al)–BDC  with a slightly smaller pore dimension (7.3 × 7.7 Å2vs. 8.5 × 8.5 Å2),IV) analogue,W = P × ΔV) absorbed during one compression–decompression cycle through an expected increase in the structural transition pressure (P) while maintaining a relatively high volume variation (ΔV).1Metal organic frameworks (MOFs) have aroused a great interest over the past decade not only for the wide spectrum of materials that can be synthesized but also for their potential use in societally-relevant applications.in situ high-pressure synchrotron X-ray powder diffraction coupled with molecular simulations confirmed that the dehydrated version of MIL-53(Al)–FA shows a reversible structural contraction  and at PSICHE beamline of the Synchrotron Soleil  using a monochromatic beam (50 × 50 μm2) with a wavelength of λ = 0.37380 Å. The pressure was generated with a membrane diamond anvil cell (MDAC) using silicon oil AP 100 (Aldrich) as the pressure-transmitting medium as its kinetic diameter largely exceeds the window size of the fumarate which ensures that it does not enter inside the pores. The applied pressure was determined from the shift of the ruby R1 fluorescence line.Powder of the aluminum fumarate metal–organic framework MIL-53–FA has been prepared following the optimized synthesis route very recently reported by Alvarez ab initio derived flexible force-field for the MOF framework using the QuickFF protocol.Molecular simulations were performed to provide a structural model of the contracted phase detected under applied pressure. This computational effort was based on a new 3.P21/c) and a unit cell volume of 985 Å3 in good agreement with the experimental value (998.0(1) Å3) obtained from XRPD pattern indexing –FA was first explored through mercury intrusion and angle dispersive XRPD. Fig. S1–1. Considering a unit cell volume of 998 Å3 for the pristine dehydrated structure, this leads to a contracted structure with a unit cell volume of ∼750 Å3 which is significantly smaller than the cell dimensions of the pressure-induced phases previously observed for MIL-53(Al)–BDC (820 Å3),3)3)vs. benzyl groups for MIL-53, as well as the resulting absence of π–π interactions.High pressure XRPD experiments further confirmed a structural change in the same range of pressure with the appearance of new Bragg peaks above 250 MPa  that areab initio derived flexible force-field for the MOF framework –FA the best porous solid reported so far for such an application (see The compression step occurs at a pressure which is significantly higher than that observed either for MIL-53(Al)–BDC (55 MPa), MIL-53(Cr)–BDC (55 MPa), or MIL-47(V)–BDC (85 MPa). This implies that the work energy stored by MIL-53(Al)–FA, that can be calculated from the pressure transition and the corresponding volume variation, attains 60 J gtion see .It is noteworthy that, unlike for MIL-53(Al) where the transition was found to be irreversible, mercury intrusion experiments further evidenced that MIL-53(Al)–FA shows a fully reversible mechanical behavior upon compression–decompression cycles with the presence of a hysteresis of about 125 MPa. This was confirmed using high pressure XRPD which revealed that the contracted version of MIL-53(Al)–FA returns to the initial form once the pressure is released . In conj–1. Both observations concur very well with the values obtained with mercury porosimetry, the lower upper pressure vs. Hg being associated with the limit of the current oil system (250 MPa) compared to the mercury set-up (400 MPa). This strongly supports that the selected silicon oil is bulky enough not to penetrate into the MOF micropores and hence this fluid can be used to allow the monitoring of the pressure-induced structural transition of MIL-53(Al)–FA.In contrast with the Hg experiment, the increase of the volume at low pressure is not present anymore as the silicone oil is a wetting fluid that can spontaneously occupy the interparticular porosity. A step for cycle 1 occurs in the pressure range 100–250 MPa and leads to a volume variation of 0.25 mL g–1, remains very high. Both features confirm the promise of this solid as a potential nano-damper. A partial loss of volume and a decrease of the transition pressure were recorded after the first compression (from 0.25 mL g–1 and 100–250 MPa for the first cycle to 0.20 mL g–1 and 72–250 MPa for the other cycles respectively) which might be due to the presence of silicon oil at the pore aperture of the MOF at the outer surface of the particles.–1, see The silicon oil compression–decompression cycle presents a hysteresis which is consistent with the Hg porosimetry and the work energy stored, 41.7 J gThe heat dissipated by the structural transition of MIL-53(Al)–FA during the first compression/decompression cycle was further assessed using calorimetry measurements. The corresponding data are reported in i.e. other MOFs and hydrophobic silica. It is also shown that after the first cycle, the heating energy (i.e. the difference between the heat of compression and decompression energy) is around –18.7 J g–1 which is significantly higher than the value obtained for MIL-53(Al)–BDC (–5 to –6 J g–1 during cycle 1).8It is shown that the compression (contraction of the structure) is exothermic while the decompression (expansion of the structure) is endothermic and this trend is consistent with that previously reported for MIL-53(Al)–BDC.U) values (–8.4 to 6.9 J g–1) which are much higher than the value previously reported for MIL-53(Al)–BDC (–3.0 to 1.0 J g–1).This suggests that a heat evacuation protocol would need to be implemented for the use of this solid as a nano-damper. Finally, 4.The aluminum fumarate MIL-53–FA or A520 represents the best porous solid reported so far for mechanical-energy related applications, by virtue of its reversible structural switching to form a more contracted phase that can be provoked by the application of a high external pressure, resulting in outstanding performances in terms of work and heat energies. This commercialized material is particularly attractive since its low-production cost, low toxicity and high stability will not be a drawback for further device development following the concept of a MOF/silicon oil system proposed in this study.Supplementary informationClick here for additional data file.