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Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (< 0.1 wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.
Assuntos
Adesão Celular , Hidrogéis , Lasers , Oligopeptídeos , Polietilenoglicóis , Impressão Tridimensional , Hidrogéis/química , Oligopeptídeos/química , Polietilenoglicóis/química , Animais , Camundongos , Fibroblastos/citologiaRESUMO
A panoply of new tools for tracking single particles and molecules has led to an explosion of experimental data, leading to novel insights into physical properties of living matter governing cellular development and function, health and disease. In this Perspective, we present tools to investigate the dynamics and mechanics of living systems from the molecular to cellular scale via single-particle techniques. In particular, we focus on methods to measure, interpret, and analyse complex data sets that are associated with forces, materials properties, transport, and emergent organisation phenomena within biological and soft-matter systems. Current approaches, challenges, and existing solutions in the associated fields are outlined in order to support the growing community of researchers at the interface of physics and the life sciences. Each section focuses not only on the general physical principles and the potential for understanding living matter, but also on details of practical data extraction and analysis, discussing limitations, interpretation, and comparison across different experimental realisations and theoretical frameworks. Particularly relevant results are introduced as examples. While this Perspective describes living matter from a physical perspective, highlighting experimental and theoretical physics techniques relevant for such systems, it is also meant to serve as a solid starting point for researchers in the life sciences interested in the implementation of biophysical methods.
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Disciplinas das Ciências Biológicas , Imagem Individual de Molécula , Biofísica , Disciplinas das Ciências Biológicas/métodosRESUMO
Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGF-ß) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-ß and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.
Assuntos
Epitélio Pigmentado da Retina , Fator A de Crescimento do Endotélio Vascular , Alginatos , Animais , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Suínos , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mammalian cells have evolved complex mechanical connections to their microenvironment, including focal adhesion clusters that physically connect the cytoskeleton and the extracellular matrix. This mechanical link is also part of the cellular machinery to transduce, sense and respond to external forces. Although methods to measure cell attachment and cellular traction forces are well established, these are not capable of quantifying force transmission through the cell body to adhesion sites. We here present a novel approach to quantify intracellular force transmission by combining microneedle shearing at the apical cell surface with traction force microscopy at the basal cell surface. The change of traction forces exerted by fibroblasts to underlying polyacrylamide substrates as a response to a known shear force exerted with a calibrated microneedle reveals that cells redistribute forces dynamically under external shearing and during sequential rupture of their adhesion sites. Our quantitative results demonstrate a transition from dipolar to monopolar traction patterns, an inhomogeneous distribution of the external shear force to the adhesion sites as well as dynamical changes in force loading prior to and after the rupture of single adhesion sites. Our strategy of combining traction force microscopy with external force application opens new perspectives for future studies of force transmission and mechanotransduction in cells.
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Mecanotransdução Celular , Tração , Animais , Adesão Celular , Fibroblastos , Mamíferos , Fenômenos Mecânicos , Mecanotransdução Celular/fisiologia , Microscopia de Força Atômica/métodosRESUMO
The fabrication of electrically conductive hydrogels is challenging as the introduction of an electrically conductive filler often changes mechanical hydrogel matrix properties. Here, we present an approach for the preparation of hydrogel composites with outstanding electrical conductivity at extremely low filler loadings (0.34 S m-1, 0.16 vol %). Exfoliated graphene and polyacrylamide are microengineered to 3D composites such that conductive graphene pathways pervade the hydrogel matrix similar to an artificial nervous system. This makes it possible to combine both the exceptional conductivity of exfoliated graphene and the adaptable mechanical properties of polyacrylamide. The demonstrated approach is highly versatile regarding porosity, filler material, as well as hydrogel system. The important difference to other approaches is that we keep the original properties of the matrix, while ensuring conductivity through graphene-coated microchannels. This novel approach of generating conductive hydrogels is very promising, with particular applications in the fields of bioelectronics and biohybrid robotics.
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Grafite , Hidrogéis , Condutividade Elétrica , PorosidadeRESUMO
We perform a detailed statistical analysis of diffusive trajectories of membrane-enclosed vesicles (vacuoles) in the supercrowded cytoplasm of living Acanthamoeba castellanii cells. From the vacuole traces recorded in the center-of-area frame of moving amoebae, we examine the statistics of the time-averaged mean-squared displacements of vacuoles, their generalized diffusion coefficients and anomalous scaling exponents, the ergodicity breaking parameter, the non-Gaussian features of displacement distributions of vacuoles, the displacement autocorrelation function, as well as the distributions of speeds and positions of vacuoles inside the amoeba cells. Our findings deliver novel insights into the internal dynamics of cellular structures in these infectious pathogens.
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Acanthamoeba castellanii/metabolismo , Movimento , Vacúolos/metabolismo , Acanthamoeba castellanii/citologia , Acanthamoeba castellanii/fisiologia , Difusão , Modelos TeóricosRESUMO
Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push-pull azobenzenes. Push-pull azobenzenes perform a high-frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single-cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion-associated genes as a result of the nanoscale "tickling" of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.
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Compostos Azo/metabolismo , Adesão Celular , Integrinas/metabolismo , Peptídeos Cíclicos/metabolismo , Animais , Compostos Azo/química , Fenômenos Biomecânicos , Linhagem Celular , Microscopia de Força Atômica/métodos , Peptídeos Cíclicos/química , Ratos , Análise de Célula Única/métodosRESUMO
Diblock copolymer micelle nanolithography (BCML) is a versatile and efficient method to cover large surface areas with hexagonally ordered arrays of metal nanoparticles, in which the nanoparticles are equally spaced. However, this method falls short of providing a controlled allocation of such regular nanoparticle arrays with specific spacing into micropatterns. We present here a quick and high-throughput method to generate quasi-hexagonal nanoparticle structures with well-defined interparticle spacing on segments of nanotopographic Si substrates. The topographic height of these segments plays a dominant role in dictating the spacing between the gold nanoparticles, as the nanoparticle arrangement is controlled by immersion forces and by their self-assembly within the segments. Our novel strategy of employing a single-step BCML routine is a highly promising method for the fabrication of regular gold nanopatterns in micropatterns for a wide range of applications.
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The integration of additive manufacturing technologies with the pyrolysis of polymeric precursors enables the design-controlled fabrication of architected 3D pyrolytic carbon (PyC) structures with complex architectural details. Despite great promise, their use in cellular interaction remains unexplored. This study pioneers the utilization of microarchitected 3D PyC structures as biocompatible scaffolds for the colonization of muscle cells in a 3D environment. PyC scaffolds are fabricated using micro-stereolithography, followed by pyrolysis. Furthermore, an innovative design strategy using revolute joints is employed to obtain novel, compliant structures of architected PyC. The pyrolysis process results in a pyrolysis temperature- and design-geometry-dependent shrinkage of up to 73%, enabling the geometrical features of microarchitected compatible with skeletal muscle cells. The stiffness of architected PyC varies with the pyrolysis temperature, with the highest value of 29.57 ± 0.78 GPa for 900 °C. The PyC scaffolds exhibit excellent biocompatibility and yield 3D cell colonization while culturing skeletal muscle C2C12 cells. They further induce good actin fiber alignment along the compliant PyC construction. However, no conclusive myogenic differentiation is observed here. Nevertheless, these results are highly promising for architected PyC scaffolds as multifunctional tissue implants and encourage more investigations in employing compliant architected PyC structures for high-performance tissue engineering applications.
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Engenharia Tecidual , Alicerces Teciduais , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Carbono , Células Musculares , Impressão TridimensionalRESUMO
Although recent studies indicate the impact of microbes on the central nervous systems and behavior, it remains unclear how the relationship between the functionality of the nervous system, behavior, and the microbiota evolved. In this work, we analyzed the eating behavior of Hydra, a host that has a simple nervous system and a low-complexity microbiota. To identify the neuronal subpopulations involved, we used a subpopulation-specific cell ablation system and calcium imaging. The role of the microbiota was uncovered by manipulating the diversity of the natural microbiota. We show that different neuronal subpopulations are functioning together to control eating behavior. Animals with a drastically reduced microbiome had severe difficulties in mouth opening due to a significantly increased level of glutamate. This could be reversed by adding a full complement of the microbiota. In summary, we provide a mechanistic explanation of how Hydra's nervous system controls eating behavior and what role microbes play in this.
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Hydra , Microbiota , Animais , Hydra/fisiologia , Sistema Nervoso , Comportamento AlimentarRESUMO
The glaucoma burden increases continuously and is estimated to affect more than 100 million people by 2040. As there is currently no cure to restore the optic nerve damage caused by glaucoma, the only controllable parameter is the intraocular pressure (IOP). In recent years, minimally invasive glaucoma surgery (MIGS) has emerged as an alternative to traditional treatments. It uses micro-sized drainage stents that are inserted through a small incision, minimizing the trauma to the tissue and reducing surgical and postoperative recovery time. However, a major challenge for MIGS devices is foreign body reaction and fibrosis, which can lead to a complete failure of the device. In this work, the antifibrotic potential of tetrapodal ZnO (t-ZnO) microparticles used as an additive is elucidated by using rat embryonic fibroblasts as a model. A simple, direct solvent-free process for the fabrication of stents with an outer diameter of 200-400 µm is presented, in which a high amount of t-ZnO particles (45-75 wt %) is mixed into polydimethylsiloxane (PDMS) and a highly viscous polymer/particle mixture is extruded. The fabricated stents possess increased elastic modulus compared to pure PDMS while remaining flexible to adapt to the curvature of an eye. In vitro experiments showed that the fibroblast cell viability was inhibited to 43 ± 3% when stents with 75 wt % t-ZnO were used. The results indicate that cell inhibiting properties can be attributed to an increased amount of protruding t-ZnO particles on the stent surface, leading to an increase in local contacts with cells and a disruption of the cell membrane. As a secondary mechanism, the released Zn ions could also contribute to the cell-inhibiting properties in the close vicinity of the stent surface. Overall, the fabrication method and the antifibrotic and mechanical properties of developed stents make them promising for application in MIGS.
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Glaucoma , Stents , Glaucoma/cirurgia , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Animais , Ratos , Óxido de ZincoRESUMO
In optical microscopy the contrast of transparent objects achieved with conventional methods is often not satisfactory, for example for the automated recognition of cells. In this paper we present a nano-optical label-free approach for contrast enhancement based on photonic crystal slabs (PCS) as the specimen holder. Quasi-guided modes inside these structures cause an intrinsic color of the PCS, which strongly depends on the wavelength and the quality factor of the optical mode. Objects on the surface of the PCS experience a significant color and intensity contrast enhancement, as they change properties of the optical modes.
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Aumento da Imagem/instrumentação , Microscopia de Contraste de Fase/instrumentação , Manejo de Espécimes/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Fótons , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
α(v)ß(3) integrin-mediated cell adhesion is crucially influenced by how far ligands are spaced apart. To evaluate the impact of local ligand density versus global ligand density of a given surface, we used synthetic micronanostructured cell environments with user-defined ligand spacing and patterns to investigate cellular adhesion. The development of stable focal adhesions, their number, and size as well as the cellular adhesion strength proved to be influenced by local more than global ligand density.
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Adesão Celular/fisiologia , Materiais Revestidos Biocompatíveis/química , Fibroblastos/fisiologia , Integrina alfaVbeta3/metabolismo , Animais , Células Cultivadas , RatosRESUMO
Hydrogels are attractive biomaterials because their chemical and mechanical properties can be tailored to mimic those of biological tissues. However, many hydrogels do not allow cell or protein attachment. Therefore, they are post-synthetically functionalized by adding functional groups for protein binding, which then allows cell adhesion in cell culture substrates. However, the degree of functionalization and covalent binding is difficult to analyze in these cases. Moreover, the density of the functional groups and the homogeneity of their distribution is hard to control. This work introduces another strategy for the biofunctionalization of hydrogels: we synthesized a polymerizable linker that serves as a direct junction between the polymeric structure and cell adhesion proteins. This maleimide-containing, polymerizable bio-linker was copolymerized with non-functionalized monomers to produce a bioactive hydrogel based on poly(2-hydroxyethyl methacrylate) (pHEMA). Therefore, the attachment site was only controlled by the polymerization process and was thus uniformly distributed throughout the hydrogel. In this way, the bio-conjugation by a protein-binding thiol-maleimide Michael-type reaction was possible in the entire hydrogel matrix. This approach enabled a straightforward and highly effective biofunctionalization of pHEMA with the adhesion protein fibronectin. The bioactivity of the materials was demonstrated by the successful adhesion of fibroblast cells.
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Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC). To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.
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Hidrogéis , Tração , Adesão Celular , Fenômenos Mecânicos , Microscopia de Força Atômica/métodosRESUMO
OBJECTIVES: To characterize human-gingival-fibroblast-(HGFs) viability, proliferation and adhesion on polymer-infiltrated-ceramic-network-(PICN), polyetheretherketone-(PEEK), hydroxyapatite-reinforced-polyetheretherketone-(HA-PEEK), polyetherketoneketone-(PEKK), as well as conventional titanium-(Ti) and zirconia ceramic-(Zr) implant materials in-vitro. METHODS: Six materials (n = 40/group, 240 specimens) were standardized for surface roughness, assessed employing water contact angle measurements (WCA) and loaded with HGFs. HGF viability and proliferation were assessed at 24 and 72 h. Cell adhesion strength was evaluated after 24 h exposure to lateral shear forces using a shaking-device at 320 and 560-rpm.and qualitatively tested by scanning-electron-microscopy-(SEM) at 3, 24 and 72 h. RESULTS: PICN demonstrated the lowest mean WCA (48.2 ± 6.3º), followed by Zr (73.8 ± 5.1º), while HA-PEEK showed the highest WCA (87.2 ± 1.5º; p ≤ 0.05). After 24 h, Zr showed the highest mean HGFs-viability rate (88 ± 14%), while PEKK showed the lowest one (78 ± 7%). At 72 h, Zr continued to show the highest HGF-viability (80 ± 6%) compared to PEKK (67.5 ± 6%) and PEEK (67%±5). SEM did not reveal differences between different materials with respect to cell attachment at 3, 24 or 72 h. At 320 rpm shaking, HGFs showed to be best attached to PICN (mean%-of-detached-cells ± SD; 26 ± 11%) and worst to PEEK (54 ± 18%). At 560 rpm shaking, Zr showed the least detached cells (32 ± 4%), while HA-PEEK revealed the highest number of detached cells (58 ± 3%; ANOVA/Tukey-post-hoc-test, differences not statistically significant). SIGNIFICANCE: Dental implant abutment materials and their wettability strongly affect HGF proliferation and adhesion properties. Although, PICN showed the best wettability properties, Zr exhibited the strongest adhesion strength at high shaking. Within the current study's limitations, Zr remains the most biocompatible abutment material.
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Materiais Dentários , Zircônio , Fibroblastos , Gengiva , Humanos , Teste de Materiais , Propriedades de Superfície , TitânioRESUMO
Combining extensive single particle tracking microscopy data of endogenous lipid granules in living fission yeast cells with analytical results we show evidence for anomalous diffusion and weak ergodicity breaking. Namely we demonstrate that at short times the granules perform subdiffusion according to the laws of continuous time random walk theory. The associated violation of ergodicity leads to a characteristic turnover between two scaling regimes of the time averaged mean squared displacement. At longer times the granule motion is consistent with fractional Brownian motion.
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Difusão , Metabolismo dos Lipídeos , Movimento , Pinças Ópticas , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Fatores de TempoRESUMO
Cells adapt and move due to chemical, physical, and mechanical cues from their microenvironment. It is therefore important to create materials that mimic human tissue physiology by surface chemistry, architecture, and dimensionality to control cells in biomedical settings. The impact of the environmental architecture is particularly relevant in the context of cancer cell metastasis, where cells migrate through small constrictions in their microenvironment to invade surrounding tissues. Here, a synthetic hydrogel scaffold with an interconnected, random, 3D microchannel network is presented that is functionalized with collagen to promote cell adhesion. It is shown that cancer cells can invade such scaffolds within days, and both the microarchitecture and stiffness of the hydrogel modulate cell invasion and nuclear dynamics of the cells. Specifically, it is found that cell migration through the microchannels is a function of hydrogel stiffness. In addition to this, it is shown that the hydrogel stiffness and confinement, influence the occurrence of nuclear envelope ruptures of cells. The tunable hydrogel microarchitecture and stiffness thus provide a novel tool to investigate cancer cell invasion as a function of the 3D microenvironment. Furthermore, the material provides a promising strategy to control cell positioning, migration, and cellular function in biological applications, such as tissue engineering.
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Hidrogéis , Engenharia Tecidual , Animais , Adesão Celular , Movimento Celular , Colágeno , Humanos , Alicerces TeciduaisRESUMO
An increasing number of experimental studies employ single particle tracking to probe the physical environment in complex systems. We here propose and discuss what we believe are new methods to analyze the time series of the particle traces, in particular, for subdiffusion phenomena. We discuss the statistical properties of mean maximal excursions (MMEs), i.e., the maximal distance covered by a test particle up to time t. Compared to traditional methods focusing on the mean-squared displacement we show that the MME analysis performs better in the determination of the anomalous diffusion exponent. We also demonstrate that combination of regular moments with moments of the MME method provides additional criteria to determine the exact physical nature of the underlying stochastic subdiffusion processes. We put the methods to test using experimental data as well as simulated time series from different models for normal and anomalous dynamics such as diffusion on fractals, continuous time random walks, and fractional Brownian motion.
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Biofísica/métodos , Algoritmos , Difusão , Fractais , Lipídeos/química , Modelos Biológicos , Conformação Molecular , Movimento (Física) , Reprodutibilidade dos Testes , Processos Estocásticos , Fatores de TempoRESUMO
In many situations, cells migrate through tiny orifices. Examples include the extravasation of immune cells from the bloodstream for fighting infections, the infiltration of cancer cells during metastasis, and the migration of human pathogens. An extremely motile and medically relevant type of human pathogen is Acanthamoeba castellanii. In the study presented here, we investigated how a combination of microparticles and microstructured interfaces controls the migration of A. castellanii trophozoites. The microinterfaces comprised well-defined micropillar arrays, and the trophozoites easily migrated through the given constrictions by adapting the shape and size of their intracellular vacuoles and by adapting intracellular motion. After feeding the trophozoite cells in microinterfaces with synthetic, stiff microparticles of various sizes and shapes, their behavior changed drastically: if the particles were smaller than the micropillar gap, migration was still possible. If the cells incorporated particles larger than the pillar gap, they could become immobilized but could also display remarkable problem-solving capabilities. For example, they turned rod-shaped microparticles such that their short axis fit through the pillar gap or they transported the particles above the structure. As migration is a crucial contribution to A. castellanii pathogenicity and is also relevant to other biological processes in microenvironments, such as cancer metastasis, our results provide an interesting strategy for controlling the migration of cells containing intracellular particles by microstructured interfaces that serve as migration-limiting environments.