RESUMEN
The mechanical properties of the cellular nucleus are extensively studied as they play a critical role in important processes, such as cell migration, gene transcription, and stem cell differentiation. While the mechanical properties of the isolated nucleus have been tested, there is a lack of measurements about the mechanical behavior of the nucleus within intact cells and specifically about the interplay of internal nuclear components with the intracellular microenvironment, because current testing methods are based on contact and only allow studying the nucleus after isolation from a cell or disruption of cytoskeleton. Here, all-optical Brillouin microscopy and 3D chemomechanical modeling are used to investigate the regulation of nuclear mechanics in physiological conditions. It is observed that the nuclear modulus can be modulated by epigenetic regulation targeting internal nuclear nanostructures such as lamin A/C and chromatin. It is also found that nuclear modulus is strongly regulated by cytoskeletal behavior through a robust mechanism conserved in different culturing conditions. Given the active role of cytoskeletal modulation in nearly all cell functions, this work will enable to reveal highly relevant mechanisms of nuclear mechanical regulations in physiological and pathological conditions.
Asunto(s)
Núcleo Celular , Citoesqueleto , Epigénesis Genética , Nanoestructuras , CitoplasmaRESUMEN
We have developed a simple and robust probe-free quantitative PCR (qPCR) assay method that can detect minor mutant alleles with a frequency as low as 0.1% in a heterogeneous sample by introducing a novel T-blocker concept to the allele-specific PCR method. Four new KRAS and BRAF mutation detection assays were developed and their performance was demonstrated by testing a large number of replicates, utilizing a customized PCR protocol. Highly efficient and specific mutant amplification in conjunction with selective wild-type suppression by the T-blocker concept enabled 0.1% detection sensitivity using the intercalating dye-based qPCR chemistry instead of more complex target-specific dye-labeled probes. Excellent consistency in sensitivity and specificity of the T-blocker assay concept was demonstrated.
Asunto(s)
Análisis Mutacional de ADN/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Alelos , Colorantes/análisis , ADN/análisis , ADN/genética , Células HeLa , Humanos , Sustancias Intercalantes/análisis , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The mechanical properties of the nucleus are closely related to many cellular functions; thus, measuring nuclear mechanical properties is crucial to our understanding of cell biomechanics and could lead to intrinsic biophysical contrast mechanisms to classify cells. Although many technologies have been developed to characterize cell stiffness, they generally require contact with the cell and thus cannot provide direct information on nuclear mechanical properties. In this work, we developed a flow cytometry technique based on an all-optical measurement to measure nuclear mechanical properties by integrating Brillouin spectroscopy with microfluidics. Brillouin spectroscopy probes the mechanical properties of material via light scattering, so it is inherently label-free, non-contact, and non-invasive. Using a measuring beam spot of submicron size, we can measure several regions within each cell as they flow, which enables us to classify cell populations based on their nuclear mechanical signatures at a throughput of â¼200 cells per hour. We show that Brillouin cytometry has sufficient sensitivity to detect physiologically-relevant changes in nuclear stiffness by probing the effect of drug-induced chromatin decondensation.
Asunto(s)
Núcleo Celular/clasificación , Núcleo Celular/ultraestructura , Citometría de Flujo/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía Fluorescente/instrumentación , Animales , Cromatina , Citometría de Flujo/instrumentación , Procesamiento de Imagen Asistido por Computador , Ratones , Células 3T3 NIH , FenotipoRESUMEN
Brillouin spectroscopy probes the mechanical properties of material by measuring the optical frequency shift induced by photon-phonon scattering interactions. In traditional configurations, Brillouin spectrometers measure only one point of the sample at a time. This results in long acquisition times for mechanical imaging of large areas. In this work, we demonstrate a parallel detection configuration where the Brillouin shift of hundreds of points in a line can be measured simultaneously. In mm-sized samples, this novel configuration effectively shortens the acquisition time of two-dimensional Brillouin imaging from hours to tens of seconds, thus making it a powerful technology for label-free mechanical characterization of tissue and biomaterials.
Asunto(s)
Microscopía Confocal/métodos , Imagen Molecular/métodos , Espectrometría de Fluorescencia , Fenómenos MecánicosRESUMEN
Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillary-bound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.
Asunto(s)
Automatización de Laboratorios , Reacción en Cadena de la Polimerasa/métodos , Humanos , Reacción en Cadena de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (≥ 10(5)) in milliliter-scale volumes (≥ 0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100-2,000 cells) in micro-scale volumes (1-20 µl). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.
Asunto(s)
Técnicas Citológicas/instrumentación , Técnicas Citológicas/métodos , Animales , Automatización/instrumentación , Automatización/métodos , Escherichia coli/citología , Escherichia coli/inmunología , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories.
Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Análisis de Secuencia de ADN/instrumentación , ADN Bacteriano/genética , Genoma Bacteriano/genética , Genoma Humano/genética , Humanos , Integración de SistemasRESUMEN
We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/µL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.
Asunto(s)
Electroforesis por Microchip/instrumentación , Biblioteca de Genes , Análisis de Secuencia de ADN/instrumentación , ADN/análisis , ADN/química , Electroforesis por Microchip/métodos , Diseño de Equipo , Humanos , Leucocitos Mononucleares/química , Límite de Detección , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normasRESUMEN
Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%±4.8%.
Asunto(s)
ADN/análisis , Infecciones/genética , Automatización de Laboratorios , Conductometría , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Infecciones/diagnóstico , Técnicas Analíticas Microfluídicas , Reproducibilidad de los ResultadosRESUMEN
We demonstrate the optical manipulation of nanoliter aqueous droplets containing surfactant or lipid molecules and immersed in an organic liquid using near-infrared light. The resulting emulsion droplets are manipulated using both the thermocapillary effect and convective fluid motion. Droplet-pair interactions induced in the emulsion upon optical initiation and control provide direct observations of the coalescence steps in intricate detail. Droplet-droplet adhesion (bilayer formation) is observed under several conditions. Selective bilayer rupture is also realized using the same infrared laser. The technique provides a novel approach to studying thin film drainage and interface stability in emulsion dynamics. The formation of stable lipid bilayers at the adhesion interface between interacting water droplets can provide an optical platform on which to build droplet-based lipid bilayer assays. The technique also has relevance to understanding and improving microfluidics applications by devising Petri dish-based droplet assays requiring no substrate fabrication.
Asunto(s)
Luz , Membrana Dobles de Lípidos/química , Alcoholes Grasos/química , Glicerofosfatos/química , Rayos Infrarrojos , Aceite Mineral/química , Fosforilcolina/química , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Agua/químicaRESUMEN
Inhalation of vasoactive gases such as carbon dioxide and oxygen can provide strong changes in tissue hemodynamics. In this report, we present a preliminary clinical study aimed at assessing the feasibility of inhalation-based contrast with near infrared continuous wave transillumination for breast imaging. We describe a method for fitting the transient absorbance that provides the wavelength dependence of the optical pathlength as parametrized by tissue oxygenation and scatter power as well as the differential changes in oxy- and deoxy-hemoglobin. We also present a principal component analysis data reduction technique to assess the dynamic response from the tissue that uses coercion to provide single temporal eigenvalues associated with both oxy- and deoxy-hemoglobin changes.
RESUMEN
We present the development and implementation of a new near infrared transillumination imaging modality for tissue imaging. Exogenous inhaled hyperoxic and hypercarbic gases are used as "vasoactive contrast agents" via the production of changes in concentration of the endogenous HbO(2) and Hb in blood. This vasoactive differential imaging method is employed to acquire data and for subsequent image analysis. Spectroscopic changes obtained from transillumination measurements on the palms of healthy volunteers demonstrate the functionality of the imaging platform. This modality is being developed to monitor suspect breast lesions in a clinical setting based on the hypothesis that the atypical tumor vascular environment will yield sufficient contrast for differential optical imaging between diseased and healthy tissue.
Asunto(s)
Dióxido de Carbono , Medios de Contraste , Rayos Infrarrojos , Dispositivos Ópticos , Oxígeno , Transiluminación/métodos , Vasos Sanguíneos/efectos de los fármacos , Diseño de Equipo , Femenino , Hemoglobinas/metabolismo , Humanos , Masculino , Concentración Osmolar , Oxihemoglobinas/metabolismo , Transiluminación/instrumentaciónRESUMEN
We report high-speed real-time PCR performed on an unmodified disposable polystyrene Petri dish. The reaction cycle relies solely on an infrared laser for heating; no conventional heater is required. Nanoliter droplets of PCR mixture as water-in-oil emulsions printed in an array format served as individual PCR microreactors. A simple contact printing technique was developed to generate a large array of uniform sized nanoliter droplets using disposable pipette tips. Printed droplets showed variation of less than 10% in volume and the oil/water/polystyrene interface formed a compact droplet microreactor approximately spherical in shape. The uniform droplet array was used to optimize the laser power required for the two heating steps of PCR, annealing/extension and melting, while the ambient conditions were at room temperature. The optical heating allows for an extremely fast heating rate due to the selective absorption of the infrared laser by PCR buffer only and not the oil or polystyrene Petri dish, allowing completion of 40 amplification cycles in approximately 6 minutes. The quantitative assay capability of the system is also presented and discussed.
Asunto(s)
Calefacción/instrumentación , Rayos Láser , Microquímica/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Nanotecnología/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Reacción en Cadena de la Polimerasa/instrumentación , Diseño Asistido por Computadora , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.
Asunto(s)
ADN/genética , Rayos Láser , Reacción en Cadena de la Polimerasa/métodos , ADN/análisis , ADN/efectos de la radiación , Diseño de Equipo , Amplificación de Genes , Calefacción/instrumentación , Rayos Infrarrojos , Luz , Técnicas Analíticas Microfluídicas/instrumentación , Miniaturización , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Temperatura , TermodinámicaRESUMEN
Chlorosomes comprise thousands of bacteriochlorophylls (BChl c, d, or e) in a closely packed structure surrounded by a lipid-protein envelope and additionally contain considerable amounts of carotenoids, quinones, and BChl a. It has been suggested that carotenoids in chlorosomes provide photoprotection by rapidly quenching triplet excited states of BChl via a triplet-triplet energy transfer mechanism that prevents energy transfer to oxygen and the formation of harmful singlet oxygen. In this work we studied triplet energy transfer kinetics and photodegradation of chlorosomes isolated from wild-type Chlorobium tepidum and from genetically modified species with different types of carotenoids and from a carotenoid-free mutant. Supporting a photoprotective function of carotenoids, carotenoid-free chlorosomes photodegrade approximately 3 times faster than wild-type chlorosomes. However, a significant fraction of the BChls forms a long-lived, triplet-like state that does not interact with carotenoids or with oxygen. We propose that these states are triplet excitons that form due to triplet-triplet interaction between the closely packed BChls. Numerical exciton simulations predict that the energy of these triplet excitons may fall below that of singlet oxygen and triplet carotenoids; this would prevent energy transfer from triplet BChl. Thus, the formation of triplet excitons in chlorosomes serves as an alternative photoprotection mechanism.
Asunto(s)
Chlorobium/química , Transferencia de Energía/efectos de la radiación , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/efectos de la radiación , Oxígeno/química , Fotoquímica/métodos , Chlorobium/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Luz , Oxígeno/efectos de la radiación , Dosis de RadiaciónRESUMEN
A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the Förster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.
Asunto(s)
ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Técnicas de Sonda Molecular/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Reacciones Falso Negativas , Transferencia Resonante de Energía de Fluorescencia/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadAsunto(s)
Clorofila/química , Clorofila/efectos de la radiación , Transferencia de Energía , Modelos Químicos , Mapeo de Interacción de Proteínas/métodos , beta Caroteno/química , beta Caroteno/efectos de la radiación , Sitios de Unión/efectos de la radiación , Clorofila/análisis , Clorofila A , Simulación por Computador , Dimerización , Relación Dosis-Respuesta en la Radiación , Luz , Modelos Moleculares , Complejos Multiproteicos/análisis , Complejos Multiproteicos/química , Complejos Multiproteicos/efectos de la radiación , Unión Proteica/efectos de la radiación , beta Caroteno/análisisRESUMEN
The cytochrome b(6)f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the cytochrome b(6)f complex unambiguously show that a Chl a molecule is an intrinsic component of the cytochrome b(6)f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within approximately 4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the cytochrome b(6)f complex, the beta-carotene is too far (> or =14 Angstroms) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20-25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the cytochrome b(6)f complex does not change orientation in its excited state.