side and forward scatter as well as emission from the excited fluorophores) in the form of emitted photons and convert these photons to an electrical signal—a photocurrent—that goes into the electronics system.The components of the optical system … The emission filters are optimized for the same probes having center wavelengths of 480, 564, and 675 nanometers with bandwidths of 40, 28, and 130 nanometers, respectively. Our results include a comparison of FRET values obtained for calmodulin over the donor excitation wavelength range as well as time based observation of FRET of an E0GFP-mCherry protein. Several further cycles of mutation, directed modification and evolutionary selection produced mCherry, which has an excitation maximum at 587 nm and and emission maximum at 610 nm (4). mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. For the same amount of fluorescence signal (e.g., green and red), excitation mCherry Dichroic Cut-On Wavelength (nm): 593.00 Dichroic Filter: #67-083. The GFP signal is brighter at 920 nm (as shown above). Key: Shaded/numbered cells – Fluorophores with shaded cells indicate that more than one Light and Filter Set might work for this fluorophore. Red fluorescence, mCherry. Similarly, the light produced by fluorochromes has a range of wavelengths. 2. mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. The excitation/emission wavelengths of photoactivated PAmCherry are 564 nm and 595 nm. However, the closer the excitation wavelength is to 495 nm, the more fluorescence will be produced. For comparison purposes, in one-photon absorption spectra (blue lines, right axis) the actual excitation wavelength is multiplied by a factor of two. The TUNE Spectral Optimization Wizard reduces the time required for users to optimize wavelengths for new fluorophores by at least 50% by automatically identifying an optimal wavelength pair for any given fluorophore. Excitation wavelength: 900 nm for SHG, GFP and 1040 nm for mCherry Objective: CFI75 Apochromat 25XC W 1300 (NA 1.10, WD 2.0) Photos courtesy of: Drs. 528 nm. Herein, we report a structure-based rational design … (a relatively high level … BD Spectrum Viewer. Along with the common name and/or acronym for each highlighter, the peak excitation (Ex) and emission (Em) wavelengths, molar extinction coefficient (EC), quantum yield (QY), relative brightness, photostability, and physiologically relevant … The excitation filter only allows a narrow band of wavelengths to pass through it, around the peak fluorophore excitation wavelength. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. Excitation Wavelength (nm): 542 - 582 Substrate: Many glass manufacturers offer the same material characteristics under different trade names. Independent excitation of PACa and mCherry in living cells and live animals The red fluorescent protein mCherry was expected to be an appropriate tag for PACa since its excitation wavelength in the green range of visible light [13] is dis-tinctfromPACa excitation by blue and UV light [14] (Figure 1A). mCherry is a basic (constitutively fluorescent) red fluorescent protein published in 2004, derived from Discosoma sp.. This model uses 1 White LED and 7 Yellow LEDs at an excitation of 575nm and includes an emission filter at 610nm. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. The dichroic beamsplitter (aka dichroic mirror or dichromatic beamsplitter) is a thin piece of coated glass set at a 45-degree angle to the optical path of the microscope. Learn More. Spanning a range of Laura Marchetti. Emission Filter: #67-036. A short summary of this paper. Download PDF. Large Stokes Shift (LSS): Stokes shift (named after George G. Stokes) is the shift in wavelength from excitation to emission. It can be excited using a 355 nm laser paired with a 450/50 nm bandpass filter, a configuration that can be found, for example, in the BD LSRFortessa™ Cell Analyzer. Microscope, software, stand, & petri dish platform: $948. Here is a summary of these wavelengths: For example, Alexa Fluor®488 , one of the most commonly used dyes, has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser, and an emission maximum at 519 nm Alexa Fluor®488 is a fluorescein derivate and has similar properties than FITC . Compared to GFP, excitation and emission properties have been shifted to longer wavelengths with excitation and emission maxima at 514 nm and 527 nm (EYFP). MCherry, the Discosoma sp. One characteristic of EYFP is its pH sensitivity. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. In the data shown here, the excitation wavelengths and absorbance measurements were made at 430, 480, 490, and 560 nm for ECFP, EGFP, mCitrine, and mCherry, respectively. Description This is the peer reviewed version of the following article: Adany, P., Johnson, C. K. and Hui, R. (2012), Fiber laser based two-photon fret measurement of calmodulin and mcherry-E0GFP proteins. Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. However, wild type GFP was less useful for the by The respective laser excitation wavelength is mentioned in their labeling. Table 1. The Dichromatic Mirror slider controls the cut-on wavelength curve position for this element, and can be toggled on or off using the check box adjacent to the slider. The mean fluorescence of mCherry individually was 4524.8 ± 5.1 compared to the mean fluorescence of mCherry mixed with sfGFP at 4693.6 ± 162.5. For example, Alexa Fluor®488 , one of the most commonly used dyes, has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser, and an emission maximum at 519 nm Alexa Fluor®488 is a fluorescein derivate and has similar properties than FITC . Figure 13. Excitation wavelength: 900 nm for SHG, GFP and 1040 nm for mCherry Objective: CFI75 Apochromat 25XC W 1300 (NA 1.10, WD 2.0) Photos courtesy of: Drs. mCherry absorbs light between 540-590 nm and emits light in the ran… In addition, we measured two-photon induced FRET in an E 0 GFP-mCherry protein construct. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. This reflects a trade-off between maximizing the excitation efficiency and maximizing the emission capture. A mCherry-PACa fusion protein was The emission maximum is chosen and only emission light at that wavelength is allowed to pass to the detector. Page 2 Today there are over 70 FPs available, of which 56 are commonly used in flow cytometry and exhibit excitation wavelengths between 355 nm and above 600 nm ( Table 1 ). Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. The components of the optical system work in concert to shine different wavelengths of light onto the cell, collect the data (i.e. Emission Filter: #67-036. Excitation Wavelength (nm): 542 - 582 Substrate: Many glass manufacturers offer the same material characteristics under different trade names. A protonated form, the predominant state, has an excitation maximum at 395 nanometers, and a less prevalent, unprotonated form that absorbs at approximately 475 nanometers. The emission spectrum was obtained at a fixed excitation wavelength of 587nm and excitation spectrum was obtained at a fixed wavelength of 610nm. The fluorescence was measured at an excitation wavelength from 570 nm and an emission wavelength of 620 nm. the photon to travel a single wavelength) in discrete amounts termed quanta and corresponds to excitation of the fluorophore from the ground state to an excited state. Each version of mCherry was expressed in E. coli with a constitutive 92 promoter/5’-UTR. Two-photon excitation (TPE) spectra of E 0 GFP and mCherry were acquired using the confocal system previously described in a range from 740 to 950 nm. Hence, some of the excitation light can be transmitted through the dichroic mirror along with the longer wavelength fluorescence emitted by the sample. This optimal wavelength is called the excitation peak. The scale bar is 1 µm. Inversely, the excitation spectrum ranging from 447 nm to 647 nm was recorded when the wavelength of emission monochromator was set to 610 nm. The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. One characteristic of EYFP is its pH sensitivity. Construction of pBADCherry The gene encoding red fluorescent protein mCherry is commercially available cloned in E. coli vector (pBAD). 32, 3531 (1993)). Of all tested structures featuring addition of βME, only two showed an absorption wavelength consistent with the experimental observations (434 nm and 419 nm calculated vs. 410 nm experimentally). At pH 6.5 EYFP has only about 50 % of its fluorescence, which is not always a disadvantage. Compared to GFP, excitation and emission properties have been shifted to longer wavelengths with excitation and emission maxima at 514 nm and 527 nm (EYFP). For three colours you can use eGFP, eBFP2, and mCherry at 780 nm. Alexa-488 and Alexa-647 work well at 780 to 800 nm. Moreover, the mCherry with an optimal excitation wavelength of 587 nm and an emission wavelength at 610 nm allowed adequate detection with filter sets provided as a standard in most microplate readers and fluorescent microscopes. AM4115T-RFYW - Designed to observe red fluorescent protein (mCherry). The excitation filter only allows a narrow band of wavelengths to pass through it, around the peak fluorophore excitation wavelength. What is the best red fluorescent protein? wavelength to sufficiently excite these fluorochromes. Appl. mCherry is the most widely used and cited red fluorescent protein owing to its fast maturity, stability, and resistance to photobleaching. 5 shows a similar result compared to the endpoint measurement from mCherry and the serial dilution. The bandwidth of all excitation filters is 20 nanometers. Two-photon absorption spectra (red symbols, left axis) are presented versus laser wavelength, used for excitation. There are a variety of FPs that can be excited by single-photon absorption at ∼ 525 nm wavelength, such as Venus, 41 DsRed, 42 and mCherry. So GCaMP6 is a favorable candidate for longer-wavelength excitation. The user labeled a retinal section with CF405M WGA (408/452nm), anti-iFlag 488, AlexaFluor647-phalloidin and the tissue is expressing mCherry. Regardless of the excitation wavelength, however, fluorescence emission has a maximum peak wavelength at 507 nanometers, although the peak is broad and not well defined. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging Masahito Yamanaka, a Kenta Saito, bNicholas I. Smith, c Yoshiyuki Arai, Kumiko Uegaki, aYasuo Yonemaru, Kentaro Mochizuki, aSatoshi Kawata, Takeharu Nagai, b and Katsumasa Fujita a, * a Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan b … Fluorescent proteins are used to tag components in the cell, so they can be studied using fluorescence spectroscopyand fluorescence microscopy. mRuby has excitation and emission maxima at 558 nanometers and 605 nanometers (similar to mCherry), respectively and is one of the brightest monomeric red fluorescent proteins yet developed. Dye combinations that work well. phore in the past required two different excitation wavelengths, 1300-nm 3PE for the green fluorophores and 1700-nm 3PE for the red fluorophores (12). The maximum mCherry excitation wavelength was used (587 nm), but the emission of mCherry was read at 615 nm, as opposed to the maximum at 609 nm. 528 nm. The maximum mCherry excitation wavelength was used (587 nm), but the emission of mCherry was read at 615 nm, as opposed to the maximum at 609 nm. The absorption wavelengths for the mCherry chromophore matched very well with the experimental observation (579 nm vs. 587 nm experimentally). The OD 600 of the cultures was measured as well as the emission of the mCherry fluorescence at a wavelength of 610 nm upon excitation at a wavelength of 587 nm. 43 Therefore, simultaneous excitation of FPs by using both single- and two-photon absorption is practical for expanding the usable emission wavelength range for FP imaging. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two‐photon excitation (TPE). Likewise, emission of a photon through fluorescence or phosphorescence is also measured in terms of quanta. These results indicate that βME converts mCherry molecules from a red fluorescent state to a blue fluorescent photoconvertible state with absorption maximum at 410 nm and fluorescence emission maximum at 460 nm. Fig. 3. βME transforms mCherry into a blue fluorescent protein. Toshiaki Mochizuki and Ichiro Masai, Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University Therefore, mVenus was excitated at 485 nm (excitation peak is at 515 nm), and the emission of mVenus was collected at its maximum position, i.e. It is, therefore, only possible to detect this fluorochrome in situations where the copy number is high. The excitation filter passes wavelengths within the mCherry excitation/absorption spectrum with a range of 542 - 582nm while the emission filter passes wavelengths within the mCherry emission spectrum with a range of 603 - 678nm. Multicolor fluorescence imaging and activity recording in the mouse brain with single-wavelength excitation. The standards were excited with the same wavelengths and fluorescein was used for ECFP, Rhodamine 6G was used for EGFP and mCitrine and Rhodamine 101 was used for mCherry. Microscope & software: $849. To prevent this unwanted light from reaching the detection system, an emission filter is used in addition to the dichroic mirror. The “short” wavelength red fluorescent proteins with the longest excitation maxima (including mCherry, mPlum, HcRed, TagFP635, etc.) The best way to selectively bleach mCherry is to bleach near its excitation wavelength (~587 nm). As demonstrated in Figure 1, the red-shifted variants, typified by EGFP, have a single excitation peak centered at about 488 nm, with an emission peak wavelength of 509 nm. Fused Silica. The spectra of PE at two different excitation wavelengths, shown in Figure 2, illustrate this phenomenon even more compellingly. Genes cloned into the MCS will be expressed as fusions to the C-terminus of PAmCherry if they are in the same reading frame as PAmCherry and there are no intervening stop codons. Combining 3PE to a higher-energy band for red fluorophores with 3PE to the lowest-energy band for green fluorophores, we demonstrate simultaneous multicolor 3PM using a single-wavelength excitation at 1340 nm. A compilation of properties of the most useful optical highlighter fluorescent protein variants is presented in Table 1. mCherry: 610nm: 587nm: mCitrine: 529nm: 514nm: MitoTracker Deep Red … If you are imaging tdTomato and GFP opt for 920 nm instead of 800 nm. 1, and the measured fluorescence intensity of mCherry is plotted against excitation wavelength in Fig. Bacteria Escherichia coli ID: 106877 Maximal excitation wavelength for dsRed fluorophore. For LSS proteins, the Stokes shift is ≥ 100nm. 3. Table 2 – Fluorophores and recommended NIGHTSEA wavelength sets. The mammalian ER was imaged by conventional confocal microscopy (left) or by STED microscopy (right) with 635 nm excitation and a STED wavelength of 760 nm. The respective laser excitation wavelength is mentioned in their labeling. The energy in a quantum (Planck's Law) is expressed by the equation: Fig. Its excitation and emission maxima occur at 554 nm and 581 nm, respectively (1). As an added bonus, the fluorescent protein retains the high Stokes shift of eqFP611 and is resistant to acidic environments (pKa of 4.4). The fluorescence measurements of the different mCherry versions are presented in 93 Figure 2. Six generations of directed evolution [of mOrange] with constant photostability selection yielded the variant mApple, which, though substantially brighter than mCherry, displayed complex photoswitching behavior.This behavior was more pronounced with continuous wide-field than with laser-scanning illumination and could be largely eliminated by excitation at alternate … How do you find an RFP? DAPI (4,6-Diamidino-2-phenylindole) is a fluorescent compound with an excitation peak at 359 nm and an emission peak at 457 nm, giving it a fairly large Stokes' Shift of 98nm. The spectral properties of the purified mCherry were measured in a fluorometer (PTI Quanta Master™). Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. Each acquisition was taken for 2–4 s with f-stop one and medium binning. PAmCherry is a photoactivatable mutant of the fluorescent protein mCherry. PAmCherry is non-fluorescence until photoactivated by a short exposure to light at a wavelength between 350 nm and 400 nm. The excitation/emission wavelengths of photoactivated PAmCherry are 564 nm and 595 nm. These excitation/emission properties were well-sui-ted to imaging applications using microscopes equipped with mer-cury arc lamps. Representative fluorescent images of tissue expressing mCherry are shown at different excitation wavelengths in Fig. In the case of tdTomato, the excitation wavelength was 535 nm and emission was collected in 20 nm increments from 540 nm to 660 nm; for mCherry the excitation wavelength was 570 nm and emission was collected in 20 nm increments from 580 nm to 680 nm. The mKO excitation filter is designed to work off-peak to minimize simultaneous excitation of mPlum. Thus, we present a practical FRET measurement system with the benefits of two-photon excitation, This paper. Wavelengths of maximum absorption (≈ excitation) and emission (for example, Absorption/Emission = 485 nm/517 nm) are the typical terms used to refer to a given fluorophore, but the whole spectrum may be important to consider. It is possible to detect mCherry on the BD FACSAria™ II since the 488-nm (blue) laser provides minimal excitation (8%) at the very edge of the excitation spectrum as shown in Figure 5. Here is a summary of these wavelengths: Download Full PDF Package. The emission of light from fluorescein, ranges from 490 nm to 630 nm, and the emission peak is approximately 520 nm. tion over a wide range of excitation wavelengths. Learn More. proteins with rapidly alternating excitation over a wide range of excitation wavelengths. Be sure to also look up the excitation and emission spectra for your dye of choice. Opt. The excitation and emission wavelength of mCherry were reported. Most strikingly, it has two excitation maxima: one at around 488 nm and one at around 561 nm. Therefore, mVenus was excitated at 485 nm (excitation peak is at 515 nm), and the emission of mVenus was collected at its maximum position, i.e. To evaluate the ability to overcome the green gap under actual imaging conditions, we tested excitation power at the point where fluorophore excitation occurs, that is, at the sample plane of the microscope. However, n … Choosing the suitable excitation wavelength Expression of two types of channelrhodopsins in two pairs of receptor neurons enables worm to be attracted by blue light and be repelled by red light. Toshiaki Mochizuki and Ichiro Masai, Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. Res. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission … This light is always of longer wavelength (more to the red) than the excitation color. For most FPs, Stokes shift is less than 50nm (often much less). Dye Absorbance Wavelength Emission Wavelength Visible color DAPI 345 455 blue Hoechst 33258 345 478 blue SYTOX blue 431 480 blue Hoechst 33342 343 483 blue YOYO-1 509 509 green SYTOX green 504 533 green TOTO 1,TO-PRO-1 509 533 green SYTOX orange 547 570 yellow Chromomycin A3 445 575 yellow Mithramycin 445 575 yellow Propidium iodide 536 617 … 90 mCherry protein, we designed three versions of mCherry (version V1, V2 and V3), with increasing N-91 terminal truncations (Figure 1). The BD Spectrum Viewer is a tool that depicts the excitation and emission curves of fluorochromes common to flow cytometry. Maturation time for mCherry Fluorescent protein. It is reported to be a very rapidly-maturing monomer with … Adany et al. Moreover, the mCherry with an optimal excitation wavelength of 587 nm and an emission wavelength at 610 nm allowed adequate detection with filter sets provided as a standard in most microplate readers and fluorescent microscopes. The excitation spectrum of a given fluorochrome is determined in a similar manner by monitoring fluorescence emission at the wavelength of maximum intensity while the fluorophore is excited through a group of consecutive wavelengths. al. the coelenterate Aequeora possessed an excitation maxima in the long ultraviolet range, with emission in the green (approximately 510 nm) [4,5]. Fused Silica. Microscopists typically use a shorter wavelength to excite a fluorophore and view the longer wavelength emitted fluorescence as an image. I have a question from a user about a strange phenomenon we haven’t seen and wondered if others have seen this. RFP can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm. Quantitative FRET Analysis With the E 0 GFP-mCherry Fluorescent Protein Pair. Emission Wavelength (nm): 603 - 678 Excitation Filter: #67-033. At pH 6.5 EYFP has only about 50 % of its fluorescence, which is not always a disadvantage. Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements. ... To further examine if the mCherry fluorescent protein … To evaluate the ability to overcome the green gap under actual imaging conditions, we tested excitation power at the point where fluorophore excitation occurs, that is, at the sam-ple plane of the microscope. Results and Discussion 3.1. The current center wavelength and bandwidth for the virtual excitation and emission filters is continuously updated and displayed in red numerals above the sliders. 11 Pages. The excitation wavelength spectrum may be a very narrow or broader band, or it may be all beyond a cutoff level. Peak excitation and emission wavelengths often vary depending on the environment in which the probe finds itself. When the wavelength of excitation monochromator was set to 587 nm, the emission spectrum ranging from 510 nm to 710 nm was recorded. mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). https://www.olympus-lifescience.com/.../primer/lightandcolor/fluoroexcitation The 3-photon-excitation peak of GCaMP6 at 1320 nm occurs below the onset wavelength of significant losses due to water absorption. Excitation and Emission spectrum of mCherry. Microsc. An accurate determination of whether two fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra, but a good rule of thumb is that both the peak excitation wavelengths and peak emission wavelength of the two proteins should be separated by 50-60 nm. Skin … The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. can benefit from even longer wavelength laser excitation. On the other hand, longer wavelength near 1300-nm and high-peak-power laser light sources has been reported as the excitation light … All spectra are presented in absolute values determined per mature chromophore. Emission Wavelength (nm): 603 - 678 Excitation Filter: #67-033. However, the dual-wavelength excitation re-quires a special excitation source and a complex optical setup. Quantitative FRET Analysis With the E 0 GFP-mCherry Fluorescent Protein Pair. DsRed-Monomer Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements. Excitation-Emission spectrum of mcherry mCherry Excitation maxima: 567nm Emission maxima: 605nm Excitation and Emission spectrum of imCherry Photochemistry and Photobiology, 2009. These can be either bandpass filters or longpass filters. In addition an extensive list of fluorescent proteins, associated excitation and emission wavelengths and suggested filters and mirror combinations is provided. Several further cycles of mutation, directed modification and evolutionary selection produced mCherry, which has an excitation maximum at 587 nm and and emission maximum at 610 nm (4). We expressed the mCherry protein sequence shown in reference 4 in bacteria, purified out the mCherry and raised this rabbit polyclonal antibody. Has anyone seen mCherry convert to green emission after 405nm excitation? It’ll depend on the flow cytometer you have access to but to best detect the mCherry signal excite with the yellow-green laser at 561 nm and detect in the PE-TexasRed channel with a 610/20 bandpass filter. At the same time, we plan to use GEM-GECO as calcium indicator to examining the neuron activation, and perform feedback control in the future. pists typically use a shorter wavelength to excite a fluorophore and view the longer wavelength emitted fluorescence as an image. Fluorescent proteins are used to tag components in the cell, so they can be studied using fluorescence spectroscopy. mCherry absorbs light between 540-590 nm and emits light in the range of 550-650 nm. Similarly, at mCherry excitation and emission, similar results were observed and there was no significant difference in fluorescence intensity (Figure 13B). This tool can be used to determine appropriate filters to detect a fluorochrome as well as fluorochrome compatibility and fluorescent spillover. The J23104 shows a higher gene expression rate than P8Prom and higher than J23114 in general. Three-photon action spectra of GCaMP6f, and the absorption coefficient of water (H2O data from Kou, et. Within groups, roughly in order of excitation wavelength (families excepted). mCherry Dichroic Cut-On Wavelength (nm): 593.00 Dichroic Filter: #67-083. Confocal microscopy of (a) bright-field, fluorescent with excitation wavelengths of (b) 488 and (c) 543 nm, and (d) merged images for A549 cells after incubation with 50 μg/mL FeMn(SiO 4 ) … Our results include a comparison of FRET values obtained for calmodulin over the donor excitation wavelength range as well as time based observation of FRET of an E0GFP-mCherry protein. As with all fluorochromes, the best results are achieved by matching the laser wavelength with the excitation maxima and range of the probe.
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