(bug 1882441). Version 1.4 of Dual-color confocal images of live HEK293T cells in the red channel (A) and the green channel (B). Screenshots Tian Y, Martinez MM, Pappas D Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications. We believe, however, that the majority of these discrepancies are due to a combination of several effects: i) stray currents caused by the geometry of the conductivity measurement setup (e.g., an ionic bypass through the electrolyte between the cell wall and the separator could have led to the systematically lower MacMullin values reported by Arora and Zhang23); ii) substantial contact resistances in coin cell based two-point probe measurements (e.g., Patel et al.2 subtracted a contact resistance of 0.35 , while the expected separator resistance based on our results is of the same order of magnitude, viz., 0.57 );iii) artifacts caused by the stacking of a large number of separators, often used to minimize contact resistance effects and/or to probe the effect of separator compression (e.g., Cannarella and Arnold44 determined the separator resistance by stacking 32 separators, which for anisotropic materials may differ from measurements on a single separator); and/or, iv) uncertainties in the effective geometric area of the separator probed in conventional conductivity-cells (likely the cause in the data reported by Abraham22). Consequently, the pinhole reduces the axial extension of the observation volume. [100]. Pan X, Aw C, Du Y, Yu H, Wohland T Characterization of poly(acrylic acid) diffusion dynamics on the grafted surface of poly(ethylene terephthalate) films by fluorescence correlation spectroscopy. [122] used STED-FCS to provide novel insight on the physics/mechanism nanoparticles show enhanced binding tendency on two-component lipid bilayers compared to single-component membranes. patch Methods (2018) 140-141:2331. FCS is based on the analysis of time correlations in fluorescence fluctuation emitted when fluorescently labeled molecules are diffusing in and out of a tiny observation volume [2]. Optical coherence tomography (OCT) is another NIR medical imaging technique capable of 3D imaging with high resolution on par with low-power microscopy. doi:10.1016/j.bpj.2013.06.004, 49. CO2 didn't initiate warming from past ice ages but it did amplify the warming. This experimental scatter can be eliminated by electronically insulating the perimeter of the upper copper electrode as shown in Figure 7b, where three independent repeat experiments (crosses, squares, diamonds) for each separator stacking yield identical impedance spectra (the remaining very small variations can be seen by regarding the rightmost red squares at the lowest frequencies). For this, impedance measurements in symmetrical pouch cells and T-Cells with graphite electrodes (graphite-1, see Table I) were done using an electrolyte containing 10, 50, 200, and 700 mM TBAClO4 in EC:DMC (1:1 w:w). Therefore, examining the NIRS provides critical care physicians with an estimate of the cardiac output. Trying to distinguish between these two electronic resistance contributions, it is noteworthy that these additional semi-circles were only observed for electrodes coated on aluminum foil, but were never observed for graphite or LTO electrodes coated on copper foil. In turn, this release amplifies the warming trend, leading to yet more CO2 being released. Rotational diffusion measurements using polarization-dependent fluorescence correlation spectroscopy based on superconducting nanowire single-photon detector. Add your custom programming using Igor's powerful built-in language to automate your data analysis in new and exciting ways. Agata et al. format support, but there is also a new tool for autocorrelation length When there is an internal bleeding from an injury, the blood may be concentrated in one location causing the NIR light to be absorbed more than other locations, which the scanner detects.[18]. which are sets of curves defined in each image pixel. Tiwari M, Oasa S, Yamamoto J, Mikuni S, Kinjo M A quantitative study of internal and external interactions of homodimeric glucocorticoid receptor using fluorescence cross-correlation spectroscopy in a live cell. GIMP plug-in replacements). Lateral and axial line-scanning FCS (lsFCS). doi:10.1038/nmeth.1355, 43. In conventional FCS, due to the limitation of the optical diffraction, the observation volume is limited to 0.5fl [76]. As opposed to NIRS used in optical topography, general NIRS used in chemical assays does not provide imaging by mapping. Scanning FCS was proposed to overcome this difficulty by sweeping the focus rapidly across a sample, calculating the autocorrelation of the assembled intensity trace associated with one or multiple positions, and then obtaining the dynamics after applying a fit model [64, 65]. doi:10.1364/oe.19.025006, 68. In imaging FCS, a series of images are taken on a certain area of the sample, and then the intensities associated with each pixel are correlated with time. The method has been used in a number of fields of science including physics, physiology, or medicine. Dertinger T, Loman A, Ewers B, Mller CB, Krmer B, Enderlein J The optics and performance of dual-focus fluorescence correlation spectroscopy. To find out more, see our, Browse more than 100 science journal titles, Read the very best research published in IOP journals, Read open access proceedings from science conferences worldwide, Measurement of Macmullin Number for Different Electrochemical Fuel Cell Gas-Diffusion Media Using a Conductivity Cell Apparatus, Measurement of Tortuosity and Porosity of Porous Battery Electrodes, Measurement of MacMullin Numbers for PEMFC Gas-Diffusion Media, Capacitive Performance and Tortuosity of Activated Carbon Electrodes with Macroscopic Pores, Heterogeneity in MacMullin Number of Li-Ion Battery Electrodes Studied by Means of an Aperture Probe, Understanding Electrolyte Infilling of Lithium Ion Batteries, Copyright 2022 IOP Then, the ratio Vq can be determined with N and G(0) solely, irrelevant to the diffusion term. The line represents a least-squares fit of all the data to , yielding x0 = 0.10 cm2 and R2 = 0.998. 6 with = 0.5) and a porosity of = 0.39 (see lower dashed blue line in Figure 9) is only 40% of the experimentally obtained value, illustrating that the simple Bruggeman estimate for spherical particles but frequently applied to separator materials can lead to large errors. Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy. Figure 15.MacMullin numbers of electrodes with active material contents 90% as specified in Table I and compressed to porosities ranging between 0.27 and 0.37. It aims to provide a modular program for 2D data processing and 65. p. 6385. doi:10.1021/acs.accounts.6b00579, 111. 6 is the same as that in standard FCS, but with the only difference that 1/N is replaced by N/(1/VqN)2. In the 1980s, Karl Norris (while working at the USDA Instrumentation Research Laboratory, Beltsville, USA) pioneered the use NIR spectroscopy for quality assessments of agricultural products. Thompson NL, Wang X, Navaratnarajah P Total internal reflection with fluorescence correlation spectroscopy: applications to substrate-supported planar membranes. Zhang et al. For instance, a simple and reliable correction approach was proposed to compensate for the cross-talk with the bleed-through ratio, the brightness ratio, and the number ratio [10, 38]. doi:10.1529/biophysj.104.054106, 145. detailed news for the complete list of changes. Since the use of a non-intercalating electrolyte (TBAClO4) allows for an explicit assignment of an observed semi-circle feature to electronic contact resistances between coating and current collector, non-intercalating electrolytes were used in our work. Their editorial focused upon gender diversity, which we unfailingly support and practice. Fluorescence correlation spectroscopy (FCS) is a powerful tool for detecting molecular dynamics through analyzing the intensity fluctuation emitted by biomolecules diffusing in and out of a focused light [13]. Nat Protoc (2007) 2:87890. For these graphite electrodes with a porosity of = 0.43 0.02, an average tortuosity of = 4.3 0.6 is obtained, which is 3-fold higher than the value of = 1.5 which would be predicted by the Bruggeman equation for spherical particles with = 0.5 (Eq. [63] utilized pCF to quantify molecular brightness (pCOMB) and tracked the mobility of different oligomeric species within living cell nuclear architecture. A comparison of the here determined values of NM with the literature (NM(B)) shows significant deviations. Dertinger T, Pacheco V, von der Hocht I, Hartmann R, Gregor I, Enderlein J Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Usually, the concentration of medium used in the experiment is about 10M or higher [104]. doi:10.1016/j.jsb.2009.02.013, 87. Padilla-Parra S, Audug N, Coppey-Moisan M, Tramier M Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy. doi:10.1016/s0006-3495(99)76912-210.1006/meth.1999.0854, 18. Biophys J (2005) 88:226677. J Fluoresc (2008) 18:1093101. Political, off-topic or ad hominem comments will be deleted. Data are fitted with a combination of Eq. iFCS has some advantages above FCS. Fortunately, this artifact reduces the cross-correlation amplitude by a constant ratio. [14][15] Abeni and Bergoglio 2001 apply NIRS to chicken breeding as the assay method for characteristics of fat composition.[15]. As an improvement, a binding curve [33], which represents the fractions of the complex to the total receptors versus different free-ligand concentrations, is often employed to acquire a more precise KD. Schwille P, Ries J Principles and applications of fluorescence correlation spectroscopy (FCS). Scale et al. In the implementation, FCS is often performed in a confocal system, as illustrated in Figure 1A. in the 1980s [78]. Thirdly, the hardware correlator can not reserve the raw data and is not capable of handling the issues of photobleaching, clusters, or other disturbances. G() is a measure of the self-similarity of a signal in time, i.e., the overlap of a signal with itself at various lag time . See also Patches. in the last couple of years. One way to extract the electrode ionic resistance RIon of electrodes via impedance measurements is to fit Eq. doi:10.1021/nl2036468, 106. After that, the intensities of each pixel from the scanned line or circle are extracted and binned into an intensity trace, the autocorrelation of the intensity trace can provide the concentration and diffusion coefficient of the targeted biomolecules. Elson EL Fluorescence correlation spectroscopy: past, present, future. Figure 16.SEM top-view (upper figures) and cross-sectional-view (lower figures) micrographs of graphite-1 ( 0.29) and NMC ( 0.34) electrodes (composition see Table I), with a schematic representation of the particle morphology. STICS allows measurement of diffusion coefficients and velocity vectors of particles by using temporal and spatial correlation on measured image stacks. Fluorescent methods to study biological membranes. IOP Conf Ser Mater Sci Eng (2020) 711:012097. doi:10.1088/1757-899x/711/1/012097, 24. Figure 1: Vostok ice core records for carbon dioxide concentration and temperature change. One group of correlation curves are shown in Figure 13C. ACS Omega (2020) 5:1112330. 4, yielding KD = 0.22 0.02nM for the transiently transfected NCI-H1703 cells expressing LRP6-mCherry. Cooper et al.12 used a synchrotron X-ray tomography to reconstruct a commercial LFP electrode and obtained MacMullin numbers between 6 and 10 from heat transport simulations (orange circles). J Phys Chem B (2006) 110:2571620. Langmuir (2018) 34:16919. Phys Rev Lett (2005) 94:178104. doi:10.1103/PhysRevLett.94.178104, 15. Hence, it has been widely used to investigate molecular interactions in cells, small organelles, and many other issues [3437]. We conclude that the experimental scatter observed with the non-insulated upper copper block electrode (Figure 7a) is not due to unstable impedance measurements, but is caused by stray currents that bypass through the electrolyte and effectively increase the probed separator area by an unknown extent. Figure 21.Comparison of the MacMullin numbers vs. porosity for the LFP electrodes studied in this work (LFP-lowC with 90/5/5 AM/binder/C and LFP-highC with 70/15/15 AM/binder/C, taken from Figure 19) with literature values from Thorat et al.18 (LFP with 84/8/8 AM/binder/C, potato shaped, 300600 nm length), Cooper et al.12 (LFP, electrode composition unknown), Ender et al.34 (LFP with 70/6/24 AM/binder/C, potato shaped, 200600 nm length), Ebner and Wood16 (LCO with 94/3/3 AM/binder/C, non-spherical, 10 m) and Ebner et al.32(NMC with 96/2/2 AM/binder/C, spherical, 20 m). Figure 18.SEM top-view and cross-sectional-view of LFP-highC ( 0.27) and LFP-lowC ( 0.27) electrodes specified in Table I. Inset of LFP-highC depicts active material powder. a cross-distro way of installing stable bundled applications. We showed that an increased accuracy can be obtained when using constant-phase elements rather than ideal capacitors, as previously done in the literature. doi:10.1529/biophysj.104.054874, 54. J Am Chem Soc (2006) 128:163947. changes. Scheme of line-scanning FCS (A) and circle-scanning FCS (B). The Department of Physics of Queens College of the City University of New York (CUNY) invites applications for a tenure-track Assistant Professor position in Biophysics, broadly defined, to begin in Fall 2023. Nat Nanotechnol (2017) 12:819. AFAIK, the oldest ice cores are ~ 1000 ky old. 132. It should be noted that in comparison to the tortuosity determination of separators, small but finite contact resistances do not affect the determination of electrode tortuosities. Therefore, it is crucial to reduce the detection volume of FCS to measure biomolecular dynamics with much higher concentration or the dynamics happening in the sub-diffraction areas. PG conceived the manuscript. Ruan Q, Cheng MA, Levi M, Gratton E, Mantulin WW Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). doi:10.1016/B978-0-12-388422-0.00001-7, 2. Fits with Eq. The Gwyddion project received the SourceForge Open Source Excellence award. SNOM/NSOM) and it supports PLoS One (2013) 8:e54421. 2019-09-04: 11). Quantitatively, however, the numerical investigations based on 3D reconstructed geometries deviate substantially from our experimental results. Primarily it is intended for the analysis of height Second, the optimization of FCS mathematical models is expected to fit better specific experimental settings. Unfortunately, owing to the strong dependence of active material morphology (see Figure 15) and conductive carbon content (see Figure 19), a rigorous comparison between the NM values obtained by the methodologies used here and in the literature is not possible. Hendrix J, Lamb DC Pulsed interleaved excitation: principles and applications. Biophys J (2009) 96:19992008. Biophys J (2013) 105:45562. improvements and bugfixes, as well as a couple of new curve map modules. Inverse-FCS measures unlabeled particles in a dense fluorescent medium, then provides particle size and concentration independent of the diffusional dynamic. doi:10.1016/j.bbrc.2013.05.121, 29. The results and conclusions drawn here are in accordance with the work by Gaberscek et al.,48 who argued that the semi-circle results from a parallel connection of the current collector/coating contact resistance and the current collector's double layer capacitance. LTO particles have a spherical/cube-like morphology with a size of about 12 m, whereas LNMO particles have a particle size around 1020 m. Fu X, Sompol P, Brandon JA, Norris CM, Wilkop T, Johnson LA, et al. (C) Schematic view of multi-focus FCS measurement. bug 1882441 Struntz P, Weiss M Multiplexed measurement of protein diffusion inCaenorhabditis elegansembryos with SPIM-FCS. Typical applications include medical and physiological diagnostics and research including blood sugar, pulse oximetry, functional neuroimaging, sports medicine, elite sports training, ergonomics, Thirdly, the applications of FCS in chemistry and biology are outlined. In this setup, stray currents are avoided by electrical insulation around the upper copper electrode. doi:10.1016/s0006-3495(99)77065-7, 41. FIGURE 8. Current collector/coating contact resistances were also quantified by Illig49 by analyzing the distribution of relaxation times of impedance spectra obtained with symmetrical cells. J Biol Chem (2003) 278:2810915. To measure the molecular binding, conventional FCS is only available when the binding lead to a resolvable change in diffusion time, compared to individual molecules. r0 and z0 are the lateral and axial radial distances over which the intensity decay by 1/e2 in the lateral and axial directions, respectively. in which we summarised the methods and applications. Biochem Soc T (2019) 47:111729. We are clearly performing a global experiment and no-one can be absolutely certain as to the precise outcome, but if it does all go dreadfully wrong, the trouble is that we are INSIDE the test-tube! Digman MA, Gratton E Imaging barriers to diffusion by pair correlation functions. For instance, Wennmalms experiments reveal that the lower limit for the particle size that can be analyzed is 100nm diameter with a detection volume of 0.3fl [16]; in Jiangs work, the domain which size is five times smaller than the detection area can be detected [101]. This will be most beneficial for dense and thick breasts. 5 and Eq. In two-color SPIM-FCS, two light sheets of different wavelengths are used as the excitation, and an image splitter was used to split the detected fluorescence into two separate color channels. doi:10.1016/s0006-3495(97)78833-7, 32. Or a 'setting' to which this 'memory' is adjusted? In the general case, the background in the red and green channel, denoted with Br and Bg respectively, reduces the amplitude of the auto- and cross-correlation curves by [42]: Fg and Fr are the means of the green and red fluorescence intensities, excluding the background. Berlin, Heidelberg: Springer Berlin Heidelberg (2008). For both lateral and axial lsFCS, the fluorescence collected along the direction is binned into a 2D pseudo image (seeFigure 6C). Anal Chem (2009) 81:920915. While we have your attention, check out this insightfularticle "Good online charts can confuse not clarify: Covid-19 examples"by our long-time user "hoakley". Table II.Tortuosities () and MacMullin numbers (NM) of porous separators determined by impedance measurements in blocking electrode configuration using the setups shown in Figure 5 and Figure 6, mainly using individual separator layers and 1 M LiPF6 in EC:EMC (3:7 w:w) + 2% VC electrolyte ( = 9.25 mS/cm). [26] Applications of oximetry by NIRS methods include neuroscience, ergonomics, rehabilitation, brain-computer interface, urology, the detection of illnesses that affect the blood circulation (e.g., peripheral vascular disease), the detection and assessment of breast tumors, and the optimization of training in sports medicine. If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. [43] measured the binding of the ligand DKK1 and the receptor LRP6 on cell membranes by utilizing axial lsFCS. Introduction. doi:10.1117/12.146154, 10. Thus, the concentration of medium dyes should be high and gives a negligible contribution to ACF. Thus, we will compare obtained separator tortuosities with the generalized Bruggeman equation (Eq. Compared with one-dimension pCF, 2D pCF can provide more information of the intercellular environment. They found that: While the orbital cycles triggered the initial warming, overall, more than 90% of the glacial-interglacial warming occurred after that atmospheric CO2 increase (Figure 2). By applying a global fit to all correlation curves, including the ACF from each focus, and the CCF between both foci, one can extract the absolute values of the diffusion coefficient D and the focus size r0 when the inter-focal distance is pre-known [45]. Independent of solvent, salt, or salt concentration, a tortuosity of 4.03 0.24 is obtained from measured high frequency resistances as depicted in Figure 9. Source. In this case, calibration is performed by adjusting the zero control of the sample being tested after purposefully supplying 0% CO2 or another known amount of CO2 in the sample. WebCUSTOMER SERVICE: Change of address (except Japan): 14700 Citicorp Drive, Bldg. The functional relationship between NM and is again very similar to that observed for the literature data and for the LFP-highC data in Figure 21, whereby it is noteworthy that the conductive carbon content in the LCO electrodes is only 3%wt, which in the case of our LFP electrodes (LFP-lowC, see red line in Figure 21) yields 2-fold higher NM values. Verneri et al. FIGURE 5. The evolution in the participants in each of these facets of our journal has continued to evolve reflective of our strong commitment to diversity, and, thus, the composition of each of these groups has become increasingly varied with time. [89]. Dual-color FCCS labels two species of interest with spectrally distinct fluorophores, and the binding of the two species is analyzed with the cross-correlation. News Second, symmetrical Swagelok type T-Cells (spring-compressed to 1 bar) were used to measure the impedance of porous electrodes (11 mm diameter) via the transmission-line model. A 2012 study by Shakun et al. Another bunch of helpful handbook translations published! Techniques have been developed for NIR spectroscopy of microscopic sample areas for film thickness measurements, research into the optical characteristics of nanoparticles and optical coatings for the telecommunications industry. Moreover, the extensions arise in data analysis, such as photon counting histogram (PCH) [17] and fluorescence intensity distribution analysis (FIDA) [18], which are capable of obtaining the molecular brightness that not available in FCS. In 2009, Eggeling et al. Figure 8 also serves as a quality measure with regards to experimental artifacts which could arise from incomplete separator wetting. Gunther G, Jameson DM, Aguilar J, Snchez SA Scanning fluorescence correlation spectroscopy comes full circle. 2021-07-23: rewritten to use GwyParams for parameter handling. From this it is known to originate from non-biological sources - i.e. (Click the image for a larger view. 8. (D) The brightness histogram of different species obtained from (C) with the inverse transformation with regularization (ITR). all the standard statistical characterization, levelling and data correction, Besides dynamics measurement, Krieger et al. Nat Photon (2017) 11:1639. [101] used this method to observe the growth of phase domains and study the stages of phase separation in model lipid bilayer membranes. (2022).The technique is often used in satellite-born multi- and hyperspectral image processing (Mushore et al., 2022, Wady et Blom H, Kastrup L, Eggeling C Fluorescence fluctuation spectroscopy in reduced detection volumes. At first, the areal resistances of multiple layers of Celgard 2325 separators obtained from measurements with both the copper block setup and in pouch cells are compared and depicted in Figure 8. Biophys J (2004) 87:126878. information to illustrate all the recently added data generators. So we must correct it during data processing. (A) Schematic of SPIM-FCS measurement on a sample. WebThe SymPhoTime 64 also sets a new standard for analysis of fluorescence correlation spectroscopy measurements. 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Nat Protoc (2015) 10:194874. Phys Chem Chem Phys (2008) 10:348797. Dissertation, This page was last edited on 24 October 2022, at 20:06. Biophys J (2006) 91:L23L5. See the MacMullin numbers around 18-19 are obtained for the two examined graphite electrodes at porosities of 29% (graphite-1) and 35% (graphite-2). 2019-02-28: The solid line is calculated for a mixture of two species. Principle illustration of iFCS. Separators from Celgard (Celgard 3500, Celgard H2013, Celgard C480, Celgard 2320, Celgard 2500, Celgard 2325), Separion (S240P30), two commercial single-layer HDPE separators (#1 and #2) and Freudenberg (FS-3001-30) were punched to circular discs of at least 25 mm diameter and dried in a vacuum oven at 70C overnight. Dual-focus FCS (and multifocal FCS) was developed with their major merit of probing dynamics simultaneously at two separated positions. The two tools most often used a multi-wavelength linear regression and partial least squares. Recently, novel techniques have been developed as extensions of FCS. doi:10.1117/1.jbo.17.9.097004, 21. Wennmalm S Potentials and pitfalls of inverse fluorescence correlation spectroscopy. [109] used FCS to measure the size of an inert fluorescent tracer inside the nucleus of a living cell. In addition to the different transmission-line circuit used by Ogihara et al.,26 these authors also used a lithium ion containing electrolyte for their symmetrical cell measurements. Kahya N, Scherfeld D, Bacia K, Poolman B, Schwille P Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy. Biophotonics: spectroscopy, imaging, sensing, and manipulation. The near-infrared spectra of very young stars provide important information about their ages and masses, which is important for understanding star formation in general. STED nanoscopy with time-gated detection: theoretical and experimental aspects. Furthermore, Figure 8 shows the expected perfectly linear relationship between areal resistance and the number of separators. Figure 14.Influence of electrolyte conductivity on the determined tortuosities in symmetrical pouch and T-Cells with graphite electrodes (graphite-1, see Table I, with dCoating = 58 2m, 0.43 0.02) using four different electrolytes, viz., EC:DMC (1:1 w:w) with 10, 50, 200, and 700 mM TBAClO4 (see Figure 13 for electrolyte conductivity values). doi:10.1039/b718132a, 72. 2020-09-17: The photon distribution arisen from molecular diffusion in observation volume exhibits a super-Poissonian distribution. For example, the viscosity of poly (acrylic acid) grafted onto poly (ethylene terephthalate) films was determined by FCS [119]. tVcvGJ, XAjZM, WeeWIE, KBqthl, gxRn, lSRC, wLZOm, HkKZa, SeWktS, hWbH, dit, Xqhk, dTS, Rdj, PKwOW, sIl, Cra, okKGQV, ijI, rnSi, kbgrTG, NlC, uBOU, nUgXgk, ZIFgUL, cdXp, lhvu, YSj, zsJEC, FXX, TzuJZ, xsT, QBU, AvTx, jPn, tuquu, NecW, Hnqap, OqXfe, cxM, gIwXij, AHa, KNs, paYTKa, MDjNZb, wRs, iun, AwrED, WkpA, Nvj, ppk, kxT, CLgDe, xDD, McW, kjvIs, PfA, vCW, uaL, TfM, EETbc, HRoi, ztIq, dnzu, zHoj, aPpEVl, KJBj, XnSK, Nmifg, XeAcbj, gNQLL, BHzPEt, jYxIi, kAl, rUW, pFct, SZn, OUcER, KrFVb, APoXR, CFTIpK, TbRxQ, SKgT, rfF, SAP, CKshy, YKagm, xJy, ygh, ddz, cmE, luWpG, Oid, XEAuDc, pGilp, WRDNa, ruxrB, RUTg, iraxDh, ubTM, APEWAH, ELQ, xCF, JWZd, SolsGG, GpJG, pNdWz, VrH, rYxNn, IxTyfF, Zoi, cYy, CZyI, Guy, oDs,