RESUMO
Accurate assessment of cell stiffness distribution is essential due to the critical role of cell mechanobiology in regulation of vital cellular processes like proliferation, adhesion, migration, and motility. Stiffness provides critical information in understanding onset and progress of various diseases, including metastasis and differentiation of cancer. Atomic force microscopy and optical trapping set the gold standard in stiffness measurements. However, their widespread use has been hampered with long processing times, unreliable contact point determination, physical damage to cells, and unsuitability for multiple cell analysis. Here, we demonstrate a simple, fast, label-free, and high-resolution technique using acoustic stimulation and holographic imaging to reconstruct stiffness maps of single cells. We used this acousto-holographic method to determine stiffness maps of HCT116 and CTC-mimicking HCT116 cells and differentiate between them. Our system would enable widespread use of whole-cell stiffness measurements in clinical and research settings for cancer studies, disease modeling, drug testing, and diagnostics.
Assuntos
Holografia , Pinças Ópticas , Estimulação Acústica , Biofísica , Diferenciação CelularRESUMO
Optical tweezers are a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein-nucleic acid interactions, and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (practical optical tweezers analysis tool). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to a worm-like chain and freely jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface, which allows researchers without programming knowledge to perform sophisticated data analysis.
Assuntos
Pinças Ópticas , Solanum tuberosum , Nanotecnologia/métodos , Dobramento de Proteína , RNARESUMO
We demonstrate an effective approach to realize active and real-time temperature monitoring around the gold nanobipyramids (AuNBPs)-labeled cancer cell under 808 nm laser irradiation by combining optical tweezers and temperature-sensitive upconversion microparticles (UCMPs). On the one hand, the aptamer-modified AuNBPs that absorb laser at 808 nm not only act as an excellent photothermal reagent but also accurately and specifically bind the target cancer cells. On the other hand, the single optically trapped NaYF4:Yb3+, Er3+ UCMPs with a 980 nm laser exhibit temperature-dependent luminescence properties, where the intensity ratio of emission 525 and 547 nm varies with the ambient temperature. Therefore, real-time temperature variation monitoring is performed by 3D manipulation of the trapped single UCMP to control its distance from the AuNBPs-labeled cancer cell while being photothermally killed. The results show distance-related thermal propagation because the temperature increase reaches as high as 10 °C at a distance of 5 µm from the cell, whereas the temperature difference drops rapidly to 5 °C when this distance increases to 15 µm. This approach shows that the photothermal conversion from AuNBPs is sufficient to kill the cancer cells, and the temperature increase can be controlled within the micrometer level at a certain period of time. Overall, we present a micrometer-size thermometer platform and provide an innovative strategy to measure temperature at the micrometer level during photothermal killing of cancer cells.
Assuntos
Luminescência , Nanopartículas/química , Pinças Ópticas , Compostos Organoáuricos/química , Fototerapia , Temperatura , Células A549 , Células Cultivadas , Érbio/química , Células HEK293 , Calefação , Humanos , Lasers , Imagem Óptica , Compostos Organoáuricos/síntese química , Fatores de Tempo , Itérbio/química , Ítrio/químicaRESUMO
Devising strategies for the controlled injection of functional nanoparticles and reagents into living cells paves the way for novel applications in nanosurgery, sensing, and drug delivery. Here, we demonstrate the light-controlled guiding and injection of plasmonic Janus nanopens into living cells. The pens are made of a gold nanoparticle attached to a dielectric alumina shaft. Balancing optical and thermophoretic forces in an optical tweezer allows single Janus nanopens to be trapped and positioned on the surface of living cells. While the optical injection process involves strong heating of the plasmonic side, the temperature of the alumina stays significantly lower, thus allowing the functionalization with fluorescently labeled, single-stranded DNA and, hence, the spatially controlled injection of genetic material with an untethered nanocarrier.
Assuntos
Óxido de Alumínio/química , DNA de Cadeia Simples/administração & dosagem , Preparações de Ação Retardada/química , Ouro/química , Nanopartículas Metálicas/química , Animais , Células CHO , Cricetulus , Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Calefação , Injeções , Luz , Pinças Ópticas , TemperaturaRESUMO
Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin's working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.
Assuntos
Cardiotônicos/farmacologia , Miosinas/efeitos dos fármacos , Ureia/análogos & derivados , Trifosfato de Adenosina , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Camundongos , Método de Monte Carlo , Pinças Ópticas , Suínos , Ureia/farmacologiaRESUMO
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins.
Assuntos
Metabolismo Energético/fisiologia , Fusão de Membrana/fisiologia , Pinças Ópticas , Dobramento de Proteína , Proteínas SNARE , Animais , Humanos , Domínios Proteicos , Proteínas SNARE/química , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMO
Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Acústica , Fenômenos Biomecânicos , Engenharia Biomédica , Separação Celular/métodos , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Humanos , Hidrodinâmica , Fenômenos do Sistema Imunitário , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Força Atômica , Neoplasias/diagnóstico , Fenômenos Ópticos , Pinças Ópticas , Pressão Osmótica , Reologia , Análise de Célula Única/métodosRESUMO
We present a single cell viability assay, based on chemical gradient microfluidics in combination with optical micromanipulation. Here, we used this combination to in situ monitor the effects of drugs and chemicals on the motility of the flagellated unicellular parasite Trypanosoma brucei; specifically, the local cell velocity and the mean squared displacement (MSD) of the cell trajectories. With our method, we are able to record in situ cell fixation by glutaraldehyde, and to quantify the critical concentration of 2-deoxy-d-glucose required to completely paralyze trypanosomes. In addition, we detected and quantified the impact on cell propulsion and energy generation at much lower 2-deoxy-d-glucose concentrations. Our microfluidics-based approach advances fast cell-based drug testing in a way that allows us to distinguish cytocidal from cytostatic drug effects, screen effective dosages, and investigate the impact on cell motility of drugs and chemicals. Using suramin, we could reveal the impact of the widely used drug on trypanosomes: suramin lowers trypanosome motility and induces cell-lysis after endocytosis.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Dispositivos Lab-On-A-Chip , Análise de Célula Única/instrumentação , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Desenho de Equipamento , Glutaral/farmacologia , Microscopia , Pinças Ópticas , Suramina/farmacologia , Fatores de TempoRESUMO
Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (â¼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) â¼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.
Assuntos
Miosinas/isolamento & purificação , Miosinas/metabolismo , Estereocílios/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Chaperonas Moleculares , Dados de Sequência Molecular , Cadeias Leves de Miosina/metabolismo , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/isolamento & purificação , Subfragmentos de Miosina/metabolismo , Miosinas/genética , Pinças Ópticas , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Células Sf9 , SpodopteraRESUMO
Tissue engineering combines biological cells and synthetic materials containing chemical signaling molecules to form scaffolds for tissue regeneration. Mesenchymal stem cells (MSCs) provide an attractive source for tissue engineering due to their versatility of multipotent differentiation. Recently, it has been recognized that both chemical and mechanical stimulations are essential mediators of adhesion and differentiation of MSCs. While significant progress has been made on the understanding of chemical regulatory factors within the extracellular matrix, the effects of mechanical stimulation exerted by nanomaterials on MSCs and the underlying mechanisms are less well-known. The present study showed that the adhesion, proliferation, and differentiation of MSCs cultured on vertically aligned silicon nanowire (SiNW) arrays were significantly different from those on flat silicon wafer and control substrates. The interactions between MSCs and the SiNW arrays caused the stem cells to preferentially differentiate toward osteocytes and chondrocytes but not adipocytes in the absence of supplementary growth factors. Our study demonstrated that Ca(2+) ion channels were transiently activated in MSCs upon mechanical stimulation, which eventually led to activation of Ras/Raf/MEK/ERK signaling cascades to regulate adhesion, proliferation, and differentiation of MSCs. The stretch-mediated transient Ca(2+) ion channel activation and cytoskeleton reorganization during stem cell-nanowire interaction may be early events of lineage-specific potentiation of MSCs in determining the fates of mesenchymal stem cells cultured on microenvironments with specific mechanical properties.
Assuntos
Canais de Cálcio/metabolismo , Técnicas de Cultura de Células , Citoesqueleto/metabolismo , Células-Tronco Mesenquimais/citologia , Nanofios/química , Silício/química , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Feminino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pinças Ópticas , Transdução de Sinais , Engenharia TecidualRESUMO
Magnetic therapy is an alternative medicine practice involving the use of magnetic fields subjected to certain parts of the body and stimulates healing from a range of health problems. In this paper, an embedded nano-antenna system using the optical spins generated from a particular configuration of microrings (PANDA) is proposed. The orthogonal solitons pairs corresponding to the left-hand and right-hand optical solitons (photons) produced from dark-bright soliton conversion can be simultaneously detected within the system at the output ports. Two possible spin states which are assigned as angular momentum of either +h or -h will be absorbed by an object whenever this set of orthogonal solitons is imparted to the object. Magnetic moments could indeed arise from the intrinsic property of spins. By controlling some important parameters of the system such as soliton input power, coupling coefficients and sizes of rings, output signals from microring resonator system can be tuned and optimized to be used as magnetic therapy array.
Assuntos
Magnetoterapia/instrumentação , Modelos Teóricos , Nanomedicina/instrumentação , Pinças Ópticas , FótonsRESUMO
Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10(-3) (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10(-3) (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.
Assuntos
Eritrócitos/citologia , Processamento de Imagem Assistida por Computador/métodos , Pinças Ópticas/normas , Células Cultivadas , Meios de Cultura/química , Dextranos , Agregação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Isoanticorpos/química , Concentração Osmolar , Papaína/farmacologia , Imunoglobulina rho(D) , Eletricidade EstáticaRESUMO
Investigations of single fragile objects manipulated by optical forces with high brilliance X-ray beams may initiate the development of new research fields such as protein crystallography in an aqueous environment. We have developed a dedicated optical tweezers setup with a compact, portable, and versatile geometry for the customary manipulation of objects for synchrotron radiation applications. Objects of a few micrometers up to a few tens of micrometers size can be trapped for extended periods of time. The selection and positioning of single objects out of a batch of many can be performed semi-automatically by software routines. The performance of the setup has been tested by wide-angle and small-angle X-ray scattering experiments on single optically trapped starch granules, using a synchrotron radiation microbeam. We demonstrate here for the first time the feasibility of microdiffraction on optically trapped protein crystals. Starch granules and insulin crystals were repeatedly raster-scanned at about 50 ms exposure/raster-point up to the complete loss of the structural order. Radiation damage in starch granules results in the appearance of low-angle scattering due to the breakdown of the polysaccharide matrix. For insulin crystals, order along the densely packed [110] direction is preferentially maintained until complete loss of long-range order.
Assuntos
Pinças Ópticas , Água/química , Cristalização , Insulina/química , Tamanho da Partícula , Espalhamento a Baixo Ângulo , Solanum tuberosum/metabolismo , Amido/química , Síncrotrons , Difração de Raios X/métodosRESUMO
For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.
Assuntos
Holografia/métodos , Lasers , Micromanipulação/métodos , Microscopia/métodos , Pinças Ópticas , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Adenocarcinoma/terapia , Linhagem Celular Tumoral , Eritrócitos/citologia , Eritrócitos/fisiologia , Holografia/instrumentação , Humanos , Processamento de Imagem Assistida por Computador , Terapia a Laser/métodos , Microfluídica , Micromanipulação/instrumentação , Microscopia/instrumentação , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Neoplasias Pancreáticas/terapia , Fototerapia/métodos , Fatores de Tempo , Raios UltravioletaRESUMO
Highly efficient fabrication of well-ordered, embedded gold nanodot matrices using diffraction mask projection laser ablation is demonstrated. These gold nanodot arrays are ideally generated onto sapphire substrates but do also form onto AlO(x) thin films, enabling the application to arbitrary bulk substrates. Well-ordered gold dots become embedded into the Al(2)O(3) substrate during the process, thus improving their mechanical stability, chemical inertness, and technological compliance. Such substrates may be useful, for example, to enhance solar-cell efficiency by surface plasmons or as convenient, biocompatible focusing elements in nearfield optical tweezers.