Dynamic Light Scattering Instrument Research Articles

Assessment of Tunable Resistive Pulse Sensing (TRPS) Technology for Particle Size Distribution in Vaccine Formulations - A Comparative Study with Dynamic Light Scattering.

A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population. Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species. The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis. It also shows how TRPS may be utilized only to large size antigens due to its limited size range. The second case study highlights the difference in the sensitivities of two DLS instruments working on the same principle. The third case study show that how TRPS can better resolve the large aggregate species compare to DLS in polydisperse samples. This analysis shows that TRPS can be used as an orthogonal technique in addition to conventional DLS based methods for more precise and in-depth characterization. Both techniques are efficient in size characterization and produce comparable results, however the choice will depend on the type of formulation and size range to be evaluated.

Raynals, an online tool for the analysis of dynamic light scattering.

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size-distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication-quality figures, is free for academic use and can be accessed online on the eSPC data-analysis platform at https://spc.embl-hamburg.de/.

Dynamic light scattering based on low-cost components

A dynamic light scattering (DLS) instrument has been assembled using low-cost components. This setup includes an inexpensive laser diode, a silicon photomultiplier including amplifier electronics, a digital oscilloscope, and a regular computer. A set of latex nanoparticles with different sizes has been used to fully characterize the instrument for multiple acquisition configurations. In addition, results were compared to those obtained by transmission electron microscopy and a commercial DLS equipment including gold and iron oxide nanoparticles. The size measurements performed with the DLS instrument resulted very robust for multiple acquisition configurations with and error typically below 1%. A very good agreement was obtained when comparing the results with the values obtained with reference techniques. Therefore, accurate and reproducible size measurements can be performed with the DLS instrument developed in this work for a wide range of nanoparticle sizes and materials while significantly reducing the cost.

Dynamic Light Scattering on Nanoparticles in Microgravity in a Drop Tower

Gravity affects colloidal dispersions via sedimentation and convection. We used dynamic light scattering (DLS) to quantify the mobility of nanoparticles on ground and in microgravity. A DLS instrument was adapted to withstand the accelerations in a drop tower, and a liquid handling set-up was connected in order to stabilize the liquid temperature and enable rapid cooling or heating. Light scattering experiments were performed in the drop tower at ZARM (Bremen, Germany) during a microgravity interval of 9.1 s and compared to measurements on ground. Particle dynamics were analyzed at constant temperature and after a rapid temperature drop using a series of DLS measurements with 1 s integration time. We observed nanoparticles with average gold core diameters of 7.8 nm and non-polar oleylamine shells that were dispersed in tetradecane and had an average hydrodynamic diameter of 21 nm. The particles did not change their diameter in the observed temperature range. The particle dynamics inferred from DLS on ground and in microgravity were in good agreement, demonstrating the possibility to perform reliable DLS measurements in a drop tower.

Dynamic light scattering based on low-cost components

Abstract A dynamic light scattering (DLS) instrument has been assembled using low-cost components. This setup includes an inexpensive laser diode, a silicon photomultiplier (SiPM) including amplifier electronics, a digital oscilloscope, and a regular PC. A set of latex nanoparticles with different sizes has been used to fully characterize the instrument for multiple acquisition configurations. In addition, results were compared to those obtained by transmission electron microscopy (TEM) and a commercial DLS equipment including gold and iron oxide nanoparticles. The size measurements performed with the DLS instrument resulted very robust for multiple acquisition configurations with and error typically below 1%. A very good agreement was obtained when comparing the results with the values obtained with reference techniques. Therefore, accurate and reproducible size measurements can be performed with the DLS instrument developed in this work for a wide range of nanoparticle sizes and materials while significantly reducing the cost.

Angle-dependent effects in DLS measurements of polydisperse particles

Dynamic light scattering (DLS) is widely used for analyzing biological polymers and colloids. Its application to nanoparticles in medicine is becoming increasingly important with the recent emergence of prominent lipid nanoparticle (LNP)-based products, such as the SARS-CoV-2 vaccines from Pfizer, Inc.-BioNTech (BNT162b2) and Moderna, Inc. (mRNA-1273). DLS plays an important role in the characterization and quality control of nanoparticle-based therapeutics and vaccines. However, most DLS instruments have a single detection angle θ, and the amplitude of the scattering vector, q, varies among them according to the relationship q = (4πn/λ 0) sin(θ/2), where λ 0 is the laser wavelength. Results for identical, polydisperse samples among instruments of varying q yield different hydrodynamic diameters, because, as particles become larger they scatter less light at higher q, so that higher-q instruments will under-sample large particles in polydisperse populations, and report higher z-average diffusion coefficients, and hence smaller effective hydrodynamic diameters than lower-q instruments. As particle size reaches the Mie regime the scattering envelope manifests angular maxima and minima, and the monotonic decrease of average size versus q is lost. The discrepancy among instruments of different q is hence fundamental, and not merely technical. This work examines results for different q-value instruments, using mixtures of monodisperse latex sphere standards, for which experimental measurements agree well with computations, and also polydisperse solutions of physically-degraded LNPs, for which results follow expected trends. Mie effects on broad unimodal populations are also considered. There is no way to predict results between two instruments with different q for samples of unknown particle size distributions. Initial analysis of the polydispersity index among different instruments shows a technical difference due to method of autocorrelation analysis, in addition to the fundamental q-effect.

Physicochemical characteristics of liposome encapsulation of stingless bees' propolis

Nutraceuticals from natural sources have shown potential new leads in functional food products. Despite a broad range of health-promoting effects, these compounds are easily oxidized and unstable, making their utilization as nutraceutical ingredients limited. In this study, the encapsulated stingless bees' propolis in liposome was prepared using soy phosphatidylcholine and cholesterol by thin-film hydration technique. Three different formulations of phosphatidylcholine composition and cholesterol prepared by weight ratio was conducted to extract high propolis encapsulation. Physicochemical changes in the result of the encapsulation process are briefly discussed using scanning electron microscopy and Fourier Transform Infrared Spectroscopy. A dynamic light-scattering instrument was used to measure the hydrodynamic diameter, polydispersity index, and zeta potential. The increment of the liposomal size was observed when the concentration of extract loaded increased. In comparing three formulations, F2 (8:1 w/w) presented the best formulation as it yielded small nanoparticles of 275.9 nm with high encapsulation efficiency (66.9%). F1 (6:1 w/w) formed large particles of liposomes with 422.8 nm, while F3 (10:1 w/w) showed low encapsulation efficiency with (by) 38.7%. The liposome encapsulation will provide an effective nanocarrier system to protect and deliver the flavonoids extracted from stingless bees' propolis.

International Transportation of Ultrafine Bubble Dispersions

Interlaboratory comparison can be effective to obtain consistent findings on ultrafine bubbles (UFBs). In this study, the stability of UFBs during international transportation was reported for the first time. We conducted stability tests in cooperation with German and Canadian institutions. UFBs were measured using two laser-based characterization techniques: particle tracking analysis (PTA) and dynamic light scattering (DLS). Acceleration and temperature during transportation were also measured. The results showed that regardless of transportation, the number concentration of UFBs decreased, while the size of UFBs increased with time. By filling the glass vial with UFB dispersion and minimizing the liquid flow in the vial, the effects of transportation were virtually eliminated. It was also suggested that the liquid flow caused by vibration during transportation may have enhanced the aggregation of UFBs. We report the results of an inter-instrumental comparison among PTA and DLS instruments using the transported UFB dispersions.

Measurement of the Translational Diffusion Coefficient and Hydrodynamic Radius of Proteins by Dynamic Light Scattering.

Diffusion is a fundamental process in biological systems that governs the molecular collisions driving biochemical reactions and membrane and transport. Measurement of the diffusion coefficient and application of the Stokes-Einstein equation produces the hydrodynamic radius, which is a commonly used gauge of particle size. Additionally, measurement of the diffusion coefficient and the sedimentation coefficient, and application of the Svedberg equation, yields the molecular weight, which is particularly useful in the characterization of very large macromolecules. Dynamic light scattering (DLS) is the most common method to measure the diffusion coefficient of macromolecules. We describe a procedure to perform DLS measurements on monomeric bovine serum albumin (BSA) purified by size-exclusion chromatography using the Zetasizer Nano S particle size analyzer. We compare several analytical methods in existing software programs to estimate the diffusion coefficient of BSA (extrapolated to water at 20°C at infinite dilution, ) and describe a statistical method to obtain 95% confidence limits of the precision of the estimates. We compare estimates to literature values obtained by diffusiometry, sedimentation velocity analytical ultracentrifugation, and other DLS instruments. The method of cumulant analysis in the program SEDFIT (www.analyticalultracentrifugation.com) produced the most precise estimate, 6.06 ± 0.07 F (1 F = 10-7 cm2 s-1), which was within the range of estimates obtained by diffusiometry or sedimentation velocity. This protocol is useful for DLS method validation and quality control.

Multiple scattering effects on intercept, size, polydispersity index, and intensity for parallel (VV) and perpendicular (VH) polarization detection in photon correlation spectroscopy

Dynamic light scattering (DLS) is well established for rapid size, polydispersity, and size distribution determination of colloidal samples. While there are limitations in size range, resolution, and concentration, the technique has found ubiquitous applications from molecules to particles. With the ease of use of today’s commercial DLS instrumentation comes an inherent danger of misinterpretation or misapplication at the borderlines of suitability. In this paper, we show how comparison of different polarization components can help ascertain the presence of unwanted multiple scattering, which can lead to false conclusions about a sample’s mean size and polydispersity. We find that the contribution of multiple scattering events effectively reduces both the measured scattering intensity and the apparent size from the autocorrelation function. The intercept of the correlation function may serve as an indicator of relative strength of single to multiple scattering. Furthermore, the abundance of single scattering events at measurement positions close to the cell wall results in an apparent increase in uniformity yielding a lower polydispersity index which is more representative of the physical system.

Characterization of the Volume-based or Number-based Size Distribution for Silica Nanoparticles Using a Unique Combination of Online Dynamic Light Scattering Having a Uni-tau Multi-bit Correlator and High-resolution Centrifugal Field-Flow Fractionation Separator.

This paper presents a study of the size distributions of colloidal nanoparticles using an online dynamic light scattering (DLS) unit with a uni-tau multi-bit correlator (UMC) combined with a centrifugal field-flow fractionation (CF3) separator. Conventionally, the FFF-UV-MALS system utilizing field-flow fractionation (FFF) combined with a UV detector and multi-angle light scattering instrument (MALS) could be used to obtain the particle size distribution of colloidal nanoparticles. Lately, DLS as a technique to measure the size distributions of colloid materials has become prevalent. However, the DLS instrument will practically measure only the large particles in a multi-modal particle mixture. Therefore, the CF3-DLS w/UMC system that was developed consisted of a CF3 unit connected to an online DLS instrument with UMC. The system could measure the volume- or number-based size distribution with highly quantitative and accurate histograms for multi-modal samples. The size distributions were validated with size distributions obtained by images of an atomic force microscope (AFM). Two types of colloidal silica nanoparticles with different distribution widths were used in this study.

A multi-channel in situ light scattering instrument utilized for monitoring protein aggregation and liquid dense cluster formation

Liquid-liquid phase separation (LLPS) phenomena have been observed in vitro as well as in vivo and came in focus of interdisciplinary research activities particularly aiming at understanding the physico-chemical pathways of LLPS and its functionality in recent years. Dynamic light scattering (DLS) has been proven to be a most efficient method to analyze macromolecular clustering in solutions and suspensions with diverse applications in life sciences, material science and biotechnology. For spatially and time-resolved investigations of LLPS, i.e. formation of liquid dense protein clusters (LDCs) and aggregation, a novel eight-channel in situ DLS instrument was designed, constructed and applied. The real time formation of LDCs of glucose isomerase (GI) and bovine pancreatic trypsin inhibitor (BPTI) under different physico-chemical conditions was investigated in situ. Complex shifts in the particle size distributions indicated growth of LDCs up to the μm size regime. Additionally, near-UV circular dichroism spectroscopy was performed to monitor the folding state of the proteins in the process of LDC formation.

On the Micromixing Behavior of a Spinning Disk Reactor for Metallic Cu Nanoparticles Production

The interest in producing copper nanoparticles is high, mainly due to their enhanced properties, a wide range of possible and different applications, and the possibility for their use in the framework of catalysis. The purpose of this study is to synthesize copper nanoparticles by chemical reduction of copper sulfate with L-ascorbic acid and sodium borohydride capable to eliminate free radicals providing an antioxidant environment promoting pure copper nuclei formation. The micromixing conditions necessary for the nanoparticles production are provided by a spinning disk reactor (SDR). Relevant operational parameters, such as the disk speed velocity and the position of the reactant injectors, will lead to different product outcomes. The latter was checked by means of a dynamic light scattering instrument (DLS). At the end, depending on the adopted operating conditions, the SDR was able to produce particles between 16 nm and 39 nm, with a particle size distribution (PSD) characterized by a narrow, monomodal plot. In comparison to the smallest particles obtained in a stirred reactor tank, that is, 132 nm, the obtained results appear to be very promising.

Interlaboratory comparison of nanoparticle size measurements between NMIJ and NIST using two different types of dynamic light scattering instruments

The question of how to relate particle sizes measured using a fixed-angle dynamic light scattering (DLS) instrument with those measured using a multi-angle DLS instrument is addressed. A series of nearly monodisperse polystyrene latex (PSL) particles with nominal diameters of 100 nm, 70 nm, 50 nm, and 30 nm were measured using two different types of DLS instruments: one owned by the National Metrology Institute of Japan (NMIJ) of the multi-angle type and the other owned by the National Institute of Standards and Technology (NIST) of the fixed-angle type. The mean particle size of the PSL particles was measured using the multi-angle-type instrument at various scattering angles and at various concentrations of particle suspension. These data were used to establish the functional dependence of the measured particle size on the scattering angle and particle concentration through the least-squares fitting method. The established function was then used to predict the mean particle sizes that would have been obtained if the same scattering angle and particle concentrations as those used at NIST had been selected at NMIJ. The mean particle sizes obtained at NIST and at NMIJ agreed quite well for all four PSL particle samples after compensating for the angle and concentration differences. The result of this study clearly demonstrates that consideration for the dependence of measured particle sizes on the scattering angle and particle concentration is crucial in intra-method comparisons of mean particle sizes obtained using DLS.

A Comparative Study of Dynamic Light and X-ray Scatterings on Micelles of Topological Polymer Amphiphiles.

Micelles were prepared in organic solvents by using three topological polymer amphiphiles: (i) cyclic poly(n-decyl glycidyl ether-block-2-(2-(2-methoxyethoxy)ethoxy)ethyl glycidyl ether) (c-PDGE-b-PTEGGE) and (ii) its linear analogue (l-PDGE-b-PTEGGE); (iii) linear poly(6-phosphorylcholinehexylthiopropyl glycidyl ether-block-n-dodecanoyl glycidyl ether) (l-PPCGE-b-PDDGE). For the individual micelle solutions, the size and distribution were determined by dynamic light scattering (DLS) and synchrotron X-ray scattering analyses. The synchrotron X-ray scattering analysis further found that c-PDGE-b-PTEGGE forms oblate ellipsoidal micelle in an ethanol/water mixture, l-PDGE-b-PTEGGE makes prolate ellipsoidal micelle in an ethanol/water mixture, and l-PPCGE-b-PDDGE forms cylindrical micelle in chloroform. This comparative study found that there are large differences in the size and distribution results extracted by DLS and X-ray scattering analyses. All possible factors to cause such large differences are discussed. Moreover, a better use of the DLS instrument with keeping its merits is proposed.

Effects of Angular Dependency of Particulate Light Scattering Intensity on Determination of Samples with Bimodal Size Distributions Using Dynamic Light Scattering Methods.

The angular dependency of light scattering intensity from differently sized particles strongly influences the apparent particle size distribution, as determined by dynamic light scattering (DLS) methods. Manufactured nanomaterials have size distributions more or less; therefore, the effect of detecting the angular dependency of the apparent size distribution by DLS is crucial. Commercial DLS instruments typically have two different types of detector angular position. The first is a detector angled at 90°, and the other is a backscattering angle detector. We therefore investigated the coverage and angular dependency when determining the relative concentrations of nanoparticles in polystyrene latex samples with a bimodal size distribution, using DLS methods both experimentally and theoretically. We used five differently sized polystyrene latex particles (one was a 70-nm nanoparticle and the others were various submicron-sized particles) in a variety of mixtures (the ratio of the difference of particle sizes ranged from approximately 2 to 7) to investigate the coverage and angular dependency of the recognition of the relative concentration ratio. In the case of size difference of approximately a factor of 2 or 3 between the two mixed particles (one was fixed at 70 nm), for DLS measurements at light scattering detector angles ranging from 60° to 150°, the homodyne photon correlation functions were approximately straight lines for mixtures of two differently sized polystyrene latex particles. The straight homodyne photon correlation functions were caused by the relatively strong light scattering from larger submicron particles masking the weaker light scattering from the smaller nanoparticles. As a result, DLS analysis could not recognize the relative concentration of nanoparticles in the mixture. In contrast to these samples, for mixtures of two differently sized polystyrene latex particles (one was 70 nm in size) with a size difference of a factor of 5, the homodyne correlation functions displayed an obvious curve for angles larger than 120°. This curve reflected an appropriate relative concentration ratio for the two differently sized polystyrene latex particles. Furthermore, for a mixture of two differently sized particles (one was again 70 nm) with size differences of a factor of 7, the homodyne correlation functions showed a clearly curved shape for detector angles larger than 90°, and yielded appropriate relative concentration ratios for the two different sizes of polystyrene latex particles. These observations were supported by theoretical investigation using Mie theory and asymmetric flow field-flow fractionation measurements with a multi-angle light scattering detector. Our investigation is crucial for achieving some degree of concordance on the determination of the size distribution of particles using DLS methods in industrial and academic fields.

Dynamic Light Scattering of Biopharmaceutics-Can Analytical Performance Be Enhanced by Laser Power?

Background: Dynamic light scattering (DLS) is an important tool to characterize colloidal systems and adequate sizing is particularly critical in the field of protein formulations. Among the different factors that can influence the measurement result, the effect of laser power has so far not been studied thoroughly. Methods: The sensitivity of a DLS instrument was first considered on a theoretical level, followed by experiments using DLS instruments, equipped with two different lasers of (nominal) 45 mW, and 100 mW, respectively. This work analyzes dilute colloidal dispersions of lysozyme as model protein. Results: Theoretical findings agreed with experiments in that only enhanced laser power of 100 mW laser allowed measuring a 0.1 mg/mL protein dispersion in a reliable manner. Results confirmed the usefulness of the presented theoretical considerations in improving a general understanding of the limiting factors in DLS. Conclusions: Laser power is a critical aspect regarding adequate colloidal analysis by DLS. Practical guidance is provided to help scientists specifically with measuring dilute samples to choose both an optimal instrument configuration as well as a robust experimental procedure.

Towards reproducible measurement of nanoparticle size using dynamic light scattering: Important controls and considerations

The characterization of nanoparticles in dispersions, in particular measuring their size and size distribution, is a prerequisite before they can be used in toxicological testing. Such characterization requires reliable methods with good reproducibility. The aim of this study was to evaluate the reproducibility, and thus the potential of Dynamic Light Scattering (DLS) for nanoparticle size determination. DLS is easy to use and well established in most nanotoxicology laboratories. However, reproducibility and in particular variability between measurements done using different instrumental setups have not been addressed systematically before. Here we performed initial experiments with rather monodisperse dispersions of spherical particles in water. Significant discrepancies in the measured distributions were obtained with different DLS instruments, especially when fitting the data using mathematical inversion methods. Significant errors can be made due to different settings being used for fitting the data. These were even more prominent when working with dilute dispersions of very small particles. Our study has identified several important points to be taken into consideration in order to overcome possible issues in measurement and analysis of nanoparticles using DLS.In practice, however, nanoparticles may have significant polydispersities and/or can be non-spherical. We extend the comparative work on spherical particles, to show how to characterize polydisperse and/or high aspect ratio particles using DLS instruments.

Multi-channel in situ dynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12.

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography-SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

Preparation of Phase-Change Nanodroplets and an Experimental Study

To investigate the vaporization threshold of perfluoropentane encapsulated lipids nanodroplets and their feasibilities in assessing radio-frequency ablation margins in vitro. The nanodroplets with lipids shell and perfluoropentane (PFP) kemel was prepared by a Homogenization/emulsion method of which the size and zeta potential were measured by dynamic light scattering instrument (DLS). The phase change of perfluoropentane (PFP) nanodroplets (PFPNDs) stimulated by temperature rise was observed with the inverted optical microscope and ultrasonography. Echo intensity of bubbles generation from nanodroplets was calculated at different temperature points to plot the temperature-intensity curve. The temperature at the margins of hyperechogenicity zones generated from radiofrequency ablation (RFA) under ultrasonic guidance in tissue-mimicking phantom were measured with a thermocouple probe. The mean diameter of PFPNDs was 479.9±18.8nm. A few bubbles were observed at 50°C for PFPNDs with the inverted optical microscope, while a mass of bubbles generated rapidly from PFPNDs at 60°C, partly coalesced and ruptured ,and most of them ruptured at a higher temperature. No bubbles were observed in the Control group. With the ultrasonography, weak echo intensity of bubbles generated from PFPND at 50°C was detected. And when temperature rose to 60°C for PFPNDs or higher, the echo intensity reached peak value. Echo intensity in the Control group was not enhanced. The hyperechogenicity zones in the tissue-mimicking phantom dispersed with PFPNDs presented as elliptic shape along with the RFA needle, and the mean temperature at margin was 60.4°C. Little hyperechogenicity zones irregularly occurred in the Control group. The prepared PFPNDs can vaporize at given temperature. PFPNDs, with the vaporization threshold closed to the temperature threshold of tissues coagulative necrosis caused by RFA, is potential to assess the radiofrequency ablation margins in real time.