Embedding Medium Research Articles

In totum deparaffinization of biological samples and re‐embedded in historesin for better diagnostic

Paraffin has been the most traditional embedding medium used in histological techniques, however it can cause several technical artifacts. On the other hand, the use of plastic resins gives a more consistent and precise support to the tissues, allowing a better perfection of the histological sections. Thus, in an attempt to retrieve samples previously embedded in paraffin, we have described this new protocol which allows the material to be deparaffinized and re-embedded into historesin (glycol methacrylate). Paraffin embedded biological materials (en bloc) were deparaffinized and re-embedded with historesin. The histological sections of the samples (in paraffin and historesin) were analyzed under light microscope and the quality of the material was compared. As expected, samples embedded in historesin showed a better quality in their morphology, even if they were previously embedded in paraffin. Therefore, this new technique proposed here allows the recovery of old materials, through reprocessing to historesin, ensuring that materials already collected that cannot be obtained again are analyzed with greater clarity and great detail.

Oxidation of ultralene and paraffin due to radiation damage after exposure to soft X-rays probed by FTIR microspectroscopy and X-ray fluorescence.

Radiation damage upon soft X-ray exposure is an important issue to be considered in soft X-ray microscopy. The work presented here is part of a more extended study on the topic and focuses on the effects of soft X-rays on paraffin, a common embedding medium for soft-tissues, and on ultralene and Si3N4 windows as sample supports. Our studies suggest that the sample environment indeed plays an important role in the radiation damage process and therefore should be carefully taken into account for the analysis and interpretation of new data. The radiation damage effects were followed over time using a combination of Fourier transform infrared (FTIR) microspectroscopy and X-ray fluorescence (XRF), and it was demonstrated that, for higher doses, an oxidation of both embedding medium and ultralene substrate takes place after the irradiated sample is exposed to air. This oxidation is reflected in a clear increase of C=O and O-H infrared bands and on the XRF oxygen maps, correlated with a decrease of the aliphatic infrared signal. The results also show that the oxidation process may affect quantitative evaluation of light element concentrations.

3D fluorescence confocal microscopy of InGaN/GaN multiple quantum well nanorods from a light absorption perspective.

A nanostructure of In0.18Ga0.82N/GaN multiple quantum well (MQW) nanorods (NRs) was fabricated using top-down etching with self-organized nickel (Ni) nanoparticles as masks on the wafer. The optical properties of In0.18Ga0.82N/GaN MQW NRs were discussed by experiment and theory from a light absorption perspective. Three-dimensional (3D) optical images of NRs were successfully obtained by confocal laser scanning microscopy (CLSM) for physical observation of the optical phenomenon of InGaN/GaN MQW NRs. Moreover, optical simulations were performed by COMSOL Multiphysics via the three-dimensional finite-element method to explore the influences of NR geometrical parameters on optical absorption. The simulated results demonstrate that the absorption of NRs is higher than that of the film due to the waveguide properties of NRs resulting from their higher refractive index than embedding medium and higher aspect ratio than bulk. In addition, an increase in the diameter results in a red-shift of the absorption peak position of In0.18Ga0.82N/GaN MQW NRs. The smaller pitch enhances the near-field coupling of the nanorods and broadens the absorption peak. These results clearly illustrate the optical properties of In0.18Ga0.82N/GaN MQW NRs from the perspective of 3D confocal laser scanning microscopy. This work is promising for the applications of III–V optoelectronic devices.

Maintenance of News Frames: How US, British and Russian News Made Sense of Unfolding Events in the Syrian Chemical Weapons Crisis

ABSTRACT Frames are indispensable tools for journalists to make sense of unfolding events, but they also constrain their perspective to most readily see what they expect to see. In this study, we examine how pre-established news frames continue to inform journalists’ framing practices despite the ongoing arrival of novel, often contravening information. Specifically, we argue that dominant frames rooted in pre-existing cultural perceptions and strategic elite frame building have the capacity to overpower an open-minded appraisal of available information. In a qualitative, diachronic analysis of US, British and Russian news coverage of the 2013 Syrian Chemical Weapons crisis, we analyze journalists’ strategies for negotiating between pre-established news frames and novel, discrepant claims and observations. We find that most claims that directly contravened existing frames were either ignored or discounted by questioning the credibility of sources. By contrast, unforeseen events effectively challenged the predictive validity of dominant frames, necessitating adaptations with often far-reaching consequences for the frame. Observed patterns were consistent across outlets, despite the different journalistic cultures and embedding media systems and political settings. Our findings illuminate the important role of journalists’ pre-established ideas, which shape their news selection and framing practices, contributing to the maintenance of existing news narratives.

Design of highly-efficient acoustic waveguide couplers using impedance-tunable transformation acoustics

In this paper, the theory of impedance-tunable transformation acoustics in the geometric-acoustics limit is proposed to design efficient two-dimensional acoustic waveguide couplers. By choosing suitable impedance functions in the original space, impedance matching between the transformation medium and the background medium becomes possible, and the reflection at the boundary is reduced. The theory can be used to enable efficient acoustic coupling between waveguides of different sizes and different embedded media. By selecting an appropriate impedance function and a tunable acoustic refractive index, the transformed medium in the coupler can become a simplified parameter medium, for which the bulk modulus is a constant. This makes the experiment substantially easier. The problem of a reduced coupling-efficiency at low frequencies (a deviation from the geometric acoustic approximation) can be mitigated by selecting a large acoustic refractive index. Our two-dimensional numerical simulations indicate that this theoretical design works very well. The method can be extended to other transformation acoustic designs including three-dimensional cases.

An Another Protocol to Make Sulfur Embedded Ultrathin Sections of Extraterrestrial Small Samples.

Another protocol to make sulfur embedded ultrathin sections was developed for STXM–XANES, AFM–IR and TEM analyses of organic materials in small extraterrestrial samples. Polymerized liquid sulfur—instead of low-viscosity liquid sulfur—is the embedding media in this protocol. Due to high viscosity of the polymerized sulfur, the embedded samples stay near the surface of polymerized liquid sulfur, which facilitates trimming of glassy sulfur and ultramicrotomy of tiny embedded samples. In addition, well-continued ribbons of ultramicrotomed sections can be obtained, which are suitable for the above mentioned analyses. Because there is no remarkable difference in Carbon XANES spectra of Murchison IOM prepared by this protocol and by the conventional protocol, this protocol gives another alternative to prepare sulfur embedded ultramicrotomed sections.

Production of L-alanyl-L-glutamine by immobilized Escherichia coli expressing amino acid ester acyltransferase.

Production of Ala-Gln by the E. coli expressing α-amino acid ester acyltransferase was a promising technical route due to its high enzyme activity, but the continuous production ability still needs to improve. Therefore, the immobilized E. coli expressing α-amino acid ester acyltransferase was applied for the continuous production of Ala-Gln. Four materials were selected as embedding medium for the whole cell entrapment of recombinant bacteria. Calcium alginate beads were found to be the most proper entrapment carrier for production of Ala-Gln. The temperature, pH, and repeatability of the immobilized cell were compared with free cells. Results showed that immobilization cell could maintain a wider range of temperature/pH and better stability than free cell (20-35°C/pH8.0-9.0, and 25°C/pH8.5, respectively). On this basis, continuous production strategy was put forward by filling the immobilized cell in the tubular reactor with multiple control conditions. The Ala-Gln by immobilization cell achieved the productivity of 2.79mg/(min*mL-CV) without intermittent time. Consequently, these findings suggest that the immobilization technique has potential applications in the production of Ala-Gln by biotechnological method. KEY POINTS: • Immobilization helps to achieve high efficiency production of Ala-Gln. • Immobilized cells have better stability than free cells. • Sodium alginate is the most suitable immobilized material.

Application of infrared microscopy and alternating least squares to the forensic analysis of automotive paint chips

AbstractTo collect infrared (IR) absorbance spectra from an automotive paint chip with an IR imaging microscope, it is a common practice to cast the paint chip in epoxy and then cross section it using a microtome to reveal the individual layers of paint. Ideally, the epoxy should present little or no spectral interference. However, the epoxy can infiltrate individual layers of the paint chip as it cures contaminating the IR spectra of the layers and impairing the accuracy of a search of each of these layers against an automotive paint library. In this study, we have demonstrated that automotive paint chips can be successfully cross sectioned without the use of embedding media. Sample preparation is easier, and more importantly, interfering peaks in the spectra due to the epoxy are eliminated. To demonstrate the advantages of this approach for sample preparation, IR image maps of four automotive paint chips that were not cast in epoxy prior to cross sectioning were collected. After each IR image was unfolded using an oblique transit to traverse the image, the spectra of the individual paint layers comprising the line map were reconstructed by alternating least squares. Comparing each recovered IR spectrum against a spectral library, we show that high quality spectral matches were obtained for spectra from the same line/model of the vehicle from which the paint sample originated. When the same paint chips were cast in epoxy prior to cross sectioning, high quality spectral matches could not always be obtained.

Uniformity of the Green operator and Eshelby tensor for hyperboloidal domains in infinite media

Since Eshelby’s (1957) result (Eshelby, JD. The determination of the elastic field of an ellipsoidal inclusion and related problems. Proc R Soc London A 1957; 421: 379–396) that ellipsoids in an infinite matrix have uniform localization tensors, all attempts to find other finite domain shapes sharing that same property have failed and valuable proofs were provided that none could exist. Since that “uniformity property” also applies to infinite cylinders and layers as limits of prolate and oblate spheroids, we examine the cases of hyperboloidal domains of which infinite cylinders and platelets also are the limits. As members of the quadric surface family, hyperboloids expectably also have uniform Eshelby tensor and Green operator when embedded in infinite media, with specific features expectable too from unboundedness and not convex curvatures. Using the Radon transform method applied by the author to various inclusion shapes, as well as to finite and infinite patterns, since the uniformity of a shape function (inverse Radon transform of the domain indicator function) implies the uniformity of the related Green operator and Eshelby tensor, we examine the shape functions of axially symmetric hyperboloids. We establish that those of the two-sheet types are uniform and those of the one-sheet types are not, an additional neck-related term carrying the non-uniformity. The Green operators are next examined in considering an isotropic embedding medium with elastic- (including dielectric-) like properties. The results regarding the operator (non) uniformity correspond to those concerning the shape functions. The established operator uniformity characteristics imply validity of all Eshelby-derived ellipsoid properties. Yet, determining the Green operator solution calls for overcoming the issue of the infinite hyperbolic planar sections (the operator finiteness), with also attention being paid to positive definiteness. Options are compared from which an obtained satisfying solution with regard to both issues raises questioning theoretical and practical points on mathematical and mechanical grounds. While further studies are in progress, some application tracks are indicated.

FDTD simulation studies on improvement of light absorption in organic solar cells by dielectric nanoparticles

In this paper, we present a systematic design and analysis of organic solar cell (OSC) by embedding dielectric nanoparticles layer at anode. Using numerical simulations, we show that there is improvement in the light absorption in the active layer of the device using nanoparticles. The nanoparticles will act as scattering medium for the incident light and hence trap the light inside the device. For the light scattered at different angles, the optical path length in the active medium is increased and this leads to more absorption of light in the active layer and thereby increase in the efficiency. The scattering efficiency and hence the absorption of solar radiation for generation of current depends on the particle size, interparticle separation and the refractive index contrast between the particles and the embedding medium. Mie theory has been used to calculate the scattering efficiency of nanoparticles. The effect on light absorption and current density of OSC due to nanoparticles has been carried out using finite difference time domain analysis. It is shown that the proposed OSC structure increases the light absorption in the active layer of the device by 40% and short circuit current density by 34%.

Imaging the Cell Morphological Response to 3D Topography and Curvature in Engineered Intestinal Tissues.

While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.

Enhancement of spectral shift of plasmon resonances in bimetallic noble metal nanoparticles in core–shell structure

Core-shell metal nanoparticles (NPs) exhibit unique optical, especially plasmonic, properties in the visible range of electromagnetic spectrum. Here we report the plasmonic properties of bimetallic core-shell spherical NPs consisting of noble metals in pair (such as Au-Ag, Ag-Cu, and Au-Cu, and their interchanging combinations) embedded in silica employing Mie theory. The core radius and shell thickness were considered in the range of 2–20 nm and 1–5 nm, respectively. In the case of varied core radius, shell thickness was kept fixed, and in the case of varied shell thickness, the core radius was kept fixed. The optical extinction spectra exhibited by these NPs have two surface plasmon resonance (SPR) absorption bands in the visible region of the electromagnetic spectrum, one corresponding to the core metal and the other corresponding to shell metal, respectively. The appearance of two absorption peaks is due to the fact that in the core-shell configuration there exists two SPR modes one at the interface between the core and shell, and other at the outer surface of the shell and embedding medium. These two SPR modes interact together and determine the shape of the extinction spectra. The position of the SPR band is found to shift as the radius or the thickness of the shell is increased. The shift of the SPR peak with high range of tunability has been observed in all the cases. Such tunability is beneficial to applications in surface-enhanced Raman scattering, fluorescence, optical sensing, bio-medicine, optoelectronic devices, and others.

An effective inflation treatment for frozen section diagnosis of small-sized lesions of the lung.

BackgroundThe accuracy of intraoperative pathological diagnosis of small-sized pulmonary nodules including ground-glass opacity (GGO) is important for the surgeon to choose a suitable surgical procedure. Diagnosis of the small-sized lesions of the lung by frozen section (FS) is very difficult for the pathologist because of limited FS technology. Here we tested an effective inflation treatment for FS to improve the diagnostic accuracy of small-sized lung lesions.MethodsThe lung specimens were derived from 113 patients who underwent the surgery at Shanghai Chest Hospital in 2018–2019. The specimens were randomly divided into two groups—uninflated or inflated with diluted embedding medium (Tissue-Tek OCT; Sakura Finetek-USA, CA). The qualities of the FSs were compared with that of corresponding permanent paraffin sections. The FS diagnoses were compared with the final pathologic diagnoses of corresponding permanent sections.ResultsOur results showed that the quality of FS of lung tissue was excellent after inflation with diluted embedding medium (1:1). The total consistency between diagnosis of inflated FS and final pathological diagnosis was 85.7%. In control group, however, the consistency was only 70.2%. When the lesions were less than 1cm, the consistency between diagnosis of inflated FS and final pathological diagnosis was 90.3%, compared to 64.9% consistency in uninflated group (P=0.014, <0.05). When the lesions’ computed tomography (CT) measurement threshold ≤−350 HU, the consistency between diagnosis of inflated FS and final pathological diagnosis was 88% compared to 73.2% consistency in uninflated group (P=0.071, >0.05). Accuracy, sensitivity and specificity were observed about 90% for adenocarcinoma in situ (AIS), whereas it is drop to more than 80% for minimally invasive adenocarcinoma (MIA) in inflated FS.ConclusionsInflation with diluted embedding medium (1:1) could make lung tissue expand well during FS. By using this method, small-sized lesions (especially less than 1 cm) could be correctly diagnosed to enable adequate surgical procedure, and evaluation of which can be easily based on the intraoperative pathological diagnosis. The small lesions especially AIS could be readily identified on FS. Therefore, this method improves the diagnostic accuracy of FSs for small-sized lung lesions, and has important practical consequences for further therapy.

A simple tissue clearing method for increasing the depth penetration of STED microscopy of fixed brain slices

STED microscopy has been adopted by many research laboratories and imaging core facilities around the world. It offers biologists in principle a much higher spatial resolution than conventional light microscopy to reveal morphological and molecular details of cells beyond the diffraction barrier. However, this advantage is oftentimes hard for STED users to attain in the presence of optical aberrations, which can severely degrade microscope performance.A classic culprit is the mismatch in refractive index between the optics of the STED microscope and the biological specimen, which normally induces spherical aberrations. Inevitably, they smear out the focal intensity distributions of the excitation and STED lasers, and this problem increases dramatically with imaging depth. This means that STED microscopy has typically been restricted to distances of under 10 µm from the cover slip. However, for many neurobiological questions it is essential to image deeper into the tissue to avoid edge artifacts and to increase data throughput.Seeking to reduce such spherical aberrations and to improve the depth penetration of STED microscopy, we examined the embedding medium CFM3, which has the same refractive index as oil and is known to have a rapid clearing effect on biological tissue. We assessed CFM3 for STED imaging beyond the immediate surface of the biological sample and compared it with the common embedding medium Mowiol, using fluorescent beads and dendritic spines in fixed brain slices as test samples.We show that CFM3 makes it possible to properly resolve dendritic spines at a depth of around 40 µm with a standard commercial STED microscope, unlike Mowiol, where the spatial resolution is severely degraded. As CFM3 provides an economical and easy way to reduce the refractive index mismatch as well as to optically clear brain tissue without distorting the micro-architecture of the tissue, we recommend its use as embedding medium for super-resolution imaging deep inside brain slices.

Enhanced Methods for Needle Biopsy and Cryopreservation of Skeletal Muscle in Older Adults.

Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults.The VACORA vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in embedding medium for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples.The VACORA Biopsy System provided as many as four skeletal muscle core samples from a single biopsy site. Biopsy samples from 12 older adults weighed an average of 147.5 ± 11 mg each and had a consistent size and shape. There were no complications, and the residual scar is less than 10 mm. The freezing method using standard tissue cassettes with direct freezing in liquid nitrogen yielded high quality cryopreserved muscle tissue suitable for histological analysis without the need for isopentane and with little to no freeze-thaw damage.These enhancements have streamlined and improved the consistency of our muscle biopsy protocol and provide sufficient high-quality sample for multi-dimensional downstream studies of human muscle in aging and disease.

Optimal geometry for plasmonic sensing with non-interacting Au nanodisk arrays.

Combining finite elements method electrodynamic simulations and cost-effective and scalable nanofabrication techniques, we carried out a systematic investigation and optimization of the sensing properties of non-interacting gold nanodisk arrays. Such plasmonic nanoarchitectures offer a very effective platform for fast and simple, label-free, optical bio- and chemical-sensing. We varied their main geometrical parameters (diameter and height) to monitor the plasmonic resonance position and to find the configurations that maximize the sensitivity to small layers of an analyte (local sensitivity) or to the variation of the refractive index of an embedding medium (bulk sensitivity). The spectral position of the plasmonic resonance can be tuned over a wide range from the visible to the near-IR region (500–1300 nm) and state-of-the-art performances can be obtained using the optimized nanodisks; we obtained local and bulk sensitivities of S0 = 11.9 RIU−1 and Sbulk = 662 nm RIU−1, respectively. Moreover, the results of the simulations are compared with the performances of experimentally synthesized non-interacting Au nanodisk arrays fabricated by combining sparse colloidal lithography and hollow mask lithography, with the parameters obtained by the sensitivity numerical optimization. An excellent agreement between the experimental and the simulated results is demonstrated, confirming that the optimization performed with the simulations is directly applicable to nanosensors realized with cost-effective methods, due to the quite large stability basin around the maximum sensitivities.

Optical surface second harmonic generation from plasmonic graphene-coated nanoshells: influence of shape, size, dielectric core and embedding medium

We study optical surface second-harmonic generation (SHG) from the plasmonic nanoshells because they can improve and enhance nonlinear optical effects. We also investigate the SHG from the surface of spherical and cylindrical core-shell nanoparticles coated with a strong nonlinear material such as graphene due to its attractive plasmonic behaviour and unique properties of the surface plasmons in graphene. We demonstrate theoretically a giant and tunable second-order harmonic radiation which enhanced through the excitation of the surface plasmon resonance, can be observed at the surface of both spherical and cylindrical nanoshells because of symmetry-breaking at interface, which makes SHG as a valuable and powerful technique for applications in sensing and surface spectroscopy. We present the surface SHG strongly depends on the particle shape and size, the dielectric of embedding medium and core, the type of metal and graphene as a coating nonlinear material.

Transient nonlinear microrheology in hydrodynamically interacting colloidal dispersions: flow cessation

Relaxation of a colloidal dispersion following flow shutoff occurs over distinct time scales, providing a useful method for identifying microscopic forces that are otherwise difficult to detect. Active microrheology facilitates such interrogation for small-scale systems such as biological cells, where a Brownian probe is driven by an external force through the dispersion and its motion is tracked to infer flow properties. An interplay between external forces, Brownian motion, hydrodynamic interactions and interparticle forces deforms the microstructure surrounding the probe, which alters probe speed. Application of the Stokes drag law relates mean probe speed to the effective viscosity of the embedding medium. We present a micromechanical theoretical model for this relationship during nonlinear transient relaxation upon removal of the external force. Upon cessation, contributions to viscosity linear in the force vanish instantly, but hydrodynamics influences subsequent relaxation, as well as the pre-cessation structure that sets the initial condition. Stronger pre-cessation flow drives faster relaxation of both structure and rheology. Non-equilibrium entropic contributions persist after shutoff, decaying over time. Hydrodynamic interactions slow this relaxation by hindering relative Brownian and interparticle displacements. The dissipation of entropically stored energy produces a deterministic force that drives deterministic probe motion even after the external force is removed, giving a natural connection to the chemical potential. Using this idea, we show that probe back-travel gives a direct measure of the osmotic pressure and the time scale over which entropically stored energy is dissipated. Modelling a range of repulsions provides a platform for particle formulation tuned to desired transient response.

Influence of water based embedding media composition on the relaxation properties of fixed tissue

BackgroundIn MRI of formalin-fixed tissue one of the problems is the dependence of tissue relaxation properties on formalin composition and composition of embedding medium (EM) used for scanning. In this study, we investigated molecular mechanisms by which the EM composition affects T2 relaxation directly and T1 relaxation indirectly. ObjectiveTo identify principal components of formaldehyde based EM and the mechanism by which they affect relaxation properties of fixed tissue. MethodsWe recorded high resolution 1H NMR spectra of common formalin fixatives at temperatures in the range of 5 °C to 45 °C. We also measured T1 and T2 relaxation times of various organs of formalin fixed (FF) zebrafish at 7 T at 21 °C and 31 °C in several EM with and without fixative or gadolinium contrast agents. ResultsWe showed that the major source of T2 variability is chemical exchange between protons from EM hydroxyls and water, mediated by the presence of phosphate ions. The exchange rate increases with temperature, formaldehyde concentration in EM and phosphate concentration in EM. Depending on which side of the coalescence the system resides, the temperature increase can lead to either shortening or prolongation of T2, or to no noticeable change at all when very close to the coalescence. Chemical exchange can be minimized by washing out from EM the fixative, the phosphate or both. ConclusionThe dependence of T2 in fixed tissue on the fixative origin and composition described in prior literature could be attributed to the phosphate buffer accelerated chemical exchange among the fixative hydroxyls and the tissue water. More consistent results in the relaxation measurements could be obtained by stricter control of the fixative composition or by scanning fixed tissue in PBS without fixative.

Potential Mechanisms for Marbling Content Differences in M. Longissimus Dorsi From Wagyu And Brahman Cattle

ObjectivesIntramuscular fat (marbling) affects consumer perceptions of meat quality. Recent studies indicated that intramuscular adipocytes are derived from fibroadipogenic progenitors (FAPs), a type of bipotent mesenchymal progenitor cells in extracellular matrix of muscle fiber. Platelet-derived growth factor receptor α (PDGFRa) is a marker in identifying FAPs. The amount of PDGFRa positive cells refers to the abundance of FAPs, a key factor determining the intramuscular adipogenic efficiency of the animal. In addition, it has been shown that both satellite cells and skeletal muscle fibers regulate the activity of FAPs. In this study, we aimed to identify some underlying mechanisms for the differences in intramuscular fat accumulation between Wagyu, a high marbling breed, and Brahman, a low marbling breed, through a comparison study.Materials and MethodsFive cattle of each breed approaching mature body composition (Wagyu born January to May 2017; Brahman two 2-yr-old and three 3-yr-old, born April 2017 and 2015) were selected for this study. Biopsy samples of M. Longissimus muscle (LM) were taken after local anesthesia between the 12th and 13th ribs. Fresh samples were frozen in isopentane chilled by liquid nitrogen immediately after trimming fat and connective tissue. Other samples were fixed for 4 h in ice-cold 4% paraformaldehyde, soaked overnight in 30% sucrose solution, and then processed in Optimal Cutting Temperature embedding medium. Frozen samples were stored at –80°C before cryosectioning (5 mm) at –25°C for immunochemical staining. Unfixed muscle tissue sections were only used for muscle fiber determinations by rinsing with 1X Tris Buffer Saline (TBS) 3 times, 5 min each, before blocking in 10% goat serum and 1% bovine serum albumin, followed by overnight incubation with primary antibodies at 4°C. Subsequent secondary antibody staining at room temperature for 1 h. TBS/T (0.3% Triton) was used for membrane permeabilization to identify satellite cells, FAPs, and basement membrane. Mounting media containing 4,6-diamidino-2-phenylindole (DAPI) was used for nucleus staining. Primary antibodies against PDGFRα, paired box 7 (PAX7), laminin, and different isoforms of skeletal muscle myosins were used to identify FAPs, satellite cells, basement membrane, and different types of muscle fiber, respectively. All the sections were visualized under a Nikon inflorescence microscope, and images were analyzed by Image J software to identify different muscle fiber types and positive signals of PDGFRα and PAX7. Minimum diameter of each muscle fiber type was the average of 30 randomly selected fibers per section. Data were analyzed with R-studio using ‘t.test ()’ function with the critical value being equal to 0.05.ResultsWagyu muscle demonstrated a greater (P = 0.01) number of FAPs compared with Brahman muscle. A trend toward a higher (P = 0.06) abundance of satellite cells in Wagyu muscle than in Brahman muscles was also identified. No differences were found in muscle fiber diameter or muscle fiber type composition between these two breeds of cattle.ConclusionThe greater number of FAP cells observed in the LM of Wagyu cattle than in Brahman cattle suggests that the higher marbling content of Wagyu meat is at least partially attributed to the more abundant adipogenic progenitor cells, which increases the capacity and efficiency of intramuscular adipogenesis in Wagyu cattle.