RESUMEN
BACKGROUND: Burn injuries are a major cause of morbidity and mortality worldwide. Cooling is widely practised as a first aid measure, but the efficacy of cooling burns in human skin has not been demonstrated. A safe, consistent, ethically acceptable model of burning and cooling in live human skin in vivo was developed, and used to quantify the effects of cooling. METHODS: Novel apparatus was manufactured to create and cool burns in women who were anaesthetized for breast reconstruction surgery using a deep inferior epigastric artery perforator flap. Burns were excised between 1 and 3 h after creation, and analysed using histopathological assessment. RESULTS: All 25 women who were approached agreed to take part in the study. There were no adverse events. Increased duration of contact led to increased burn depth, with a contact time of 7·5 s at 70°C leading to a mid-dermal burn. Burn depth progressed over time following injury, but importantly this was modified by cooling the burn at 16°C for 20 min. On average, cooling salvaged 25·2 per cent of the dermal thickness. CONCLUSION: This study demonstrated the favourable effects of cooling on human burns. Public heath messaging should emphasize cooling as first aid for burns. This model will allow analysis of the molecular effects of cooling burns, and provide a platform for testing novel therapies aimed at reducing the impact of burn injury.
ANTECEDENTES: Las lesiones por quemadura son una causa importante de morbilidad y mortalidad en todo el mundo. El enfriamiento de las quemaduras se practica ampliamente como medida de primeros auxilios, pero no se ha demostrado su eficacia en la piel de los seres humanos. Para cuantificar los efectos del enfriamiento, se desarrolló un modelo de quemadura y enfriamiento en piel humana in vivo, seguro, estable y éticamente aceptable. MÉTODOS: Se construyó un dispositivo nuevo para crear y enfriar quemaduras en pacientes que fueron anestesiadas para una reconstrucción mamaria utilizando un colgajo perforante de la arteria epigástrica inferior profunda. Las quemaduras se extirparon entre una y tres horas después de su producción y se analizaron por evaluación histopatológica. Para determinar la significación estadística entre grupos se utilizó las pruebas de ANOVA o de t pareadas, según correspondiera. RESULTADOS: Aceptaron participar en el estudio las 25 pacientes a las que se propuso. No hubo efectos adversos. La mayor duración del contacto conllevó un aumento en la profundidad de la quemadura: con un tiempo de contacto de 7,5 segundos a 70°C se obtuvo una quemadura dérmica de segundo grado. La profundidad de la quemadura aumentó con el tiempo de exposición, pero mejoró de forma sustancial al enfriar la quemadura a 16°C durante 20 minutos. El enfriamiento salvó el 25% del espesor dérmico como promedio. CONCLUSIÓN: Este es el primer estudio que demuestra los efectos favorables del enfriamiento sobre las quemaduras humanas. Los consejos de salud deberían hacer más énfasis en el enfriamiento como forma de primeros auxilios en las quemaduras. Este modelo permitirá identificar los efectos moleculares del enfriamiento en las quemaduras y proporcionará una plataforma para probar nuevos tratamientos encaminados a reducir el impacto de las lesiones por quemadura.
Asunto(s)
Quemaduras/terapia , Primeros Auxilios/métodos , Hipotermia Inducida/métodos , Adulto , Quemaduras/patología , Diseño de Equipo , Femenino , Humanos , Hipotermia Inducida/instrumentación , Mamoplastia/efectos adversos , Persona de Mediana Edad , Modelos Biológicos , Colgajo PerforanteRESUMEN
BACKGROUND: The aim of this study was to demonstrate highlighting of the urethra during surgery through the use of two different methods: a new near-infrared fluorophore IRDye800BK, and indocyanine green (ICG) mixed with silicone. METHODS: Male cadavers from the department of anatomy at the University of Oxford were used to visualise the urethra during near-infrared fluorescence excitation. To assess IRDye800BK, a perineal incision was utilised after infiltrating the urethra directly with an IRDye800BK solution mixed with Instillagel. ICG-silicone was assessed when the urethra was purposely exposed as part of a simulated transanal total mesorectal dissection. ICG was previously mixed with ethanol and silicone and left to set in a Foley catheter. Fluorescence was visualised using an in-house manufactured fluorescence-enabled laparoscopic system. RESULTS: IRDye800BK demonstrated excellent penetration and visualisation of the urethra under fluorescence at an estimated tissue depth of 2 cm. An ICG-silicone catheter demonstrated excellent fluorescence without leaving any residual solution behind in the urethra after its removal. CONCLUSIONS: The newly described ICG-silicone method opens up the possibility of new technologies in this area of fluorescence guided surgery. IRDye800BK is a promising alternative to ICG in visualising the urethra using fluorescence imaging. Its greater depth of penetration may allow earlier detection of the urethra during surgery and prevent wrong plane surgery sooner.
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Colorantes Fluorescentes , Verde de Indocianina , Cuidados Intraoperatorios/métodos , Imagen Óptica/métodos , Uretra/diagnóstico por imagen , Cadáver , Fluorescencia , Humanos , Rayos Infrarrojos , Masculino , Recto/cirugía , Siliconas , Uretra/cirugíaRESUMEN
La integración de tecnologías de imagen médica en los enfoques diagnósticos y terapéuticos puede proporcionar una perspectiva preoperatoria tanto en los aspectos anatómicos (tomografía computarizada, resonancia magnética o ecografía) como funcional (tomografía computarizada de emisión de fotón único, tomografía por emisión de positrones, linfogammagrafía o imagen óptica). Además, algunas modalidades de imagen se utilizan también en un entorno intervencionista (tomografía computarizada, ecografía, imágenes gammagráficas o imágenes ópticas), donde proporcionan al cirujano información en tiempo real durante el procedimiento. En la actualidad son factibles diversas herramientas y enfoques metodológicos para la navegación guiada por imágenes en la cirugía del cáncer. Con el desarrollo de nuevos trazadores y dispositivos portátiles de imagen, estos avances reforzarán el papel de la imagen molecular intervencionista (AU)
The integration of medical imaging technologies into diagnostic and therapeutic approaches can provide a preoperative insight into both anatomical (e.g. using computed tomography, magnetic resonance imaging, or ultrasound), as well as functional aspects (e.g. using single photon emission computed tomography, positron emission tomography, lymphoscintigraphy, or optical imaging). Moreover, some imaging modalities are also used in an interventional setting (e.g. computed tomography, ultrasound, gamma or optical imaging) where they provide the surgeon with real-time information during the procedure. Various tools and approaches for image-guided navigation in cancer surgery are becoming feasible today. With the development of new tracers and portable imaging devices, these advances will reinforce the role of interventional molecular imaging (AU)
Asunto(s)
Humanos , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Cirugía Asistida por Computador/métodos , Biopsia Guiada por Imagen/métodos , Neoplasias/cirugía , Trazadores Radiactivos , Imagen Molecular/métodos , Radiofármacos/uso terapéutico , Imagen Óptica/métodos , Biopsia del Ganglio Linfático Centinela/métodosRESUMEN
The integration of medical imaging technologies into diagnostic and therapeutic approaches can provide a preoperative insight into both anatomical (e.g. using computed tomography, magnetic resonance imaging, or ultrasound), as well as functional aspects (e.g. using single photon emission computed tomography, positron emission tomography, lymphoscintigraphy, or optical imaging). Moreover, some imaging modalities are also used in an interventional setting (e.g. computed tomography, ultrasound, gamma or optical imaging) where they provide the surgeon with real-time information during the procedure. Various tools and approaches for image-guided navigation in cancer surgery are becoming feasible today. With the development of new tracers and portable imaging devices, these advances will reinforce the role of interventional molecular imaging.
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Invenciones , Neoplasias/diagnóstico por imagen , Radiografía Intervencional/métodos , Cirugía Asistida por Computador/métodos , Sistemas de Computación , Femenino , Colorantes Fluorescentes/análisis , Humanos , Laparoscopía , Mediciones Luminiscentes , Masculino , Imagen Multimodal , Metástasis de la Neoplasia , Neoplasias/cirugía , Cuidados Preoperatorios , Radiografía Intervencional/tendencias , Radiofármacos , Procedimientos Quirúrgicos Robotizados , Biopsia del Ganglio Linfático Centinela , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Cirugía Asistida por Computador/tendenciasRESUMEN
The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the "no movement" period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animal's chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.
Asunto(s)
Radioterapia Guiada por Imagen/instrumentación , Radioterapia Guiada por Imagen/veterinaria , Técnicas de Imagen Sincronizada Respiratorias/instrumentación , Técnicas de Imagen Sincronizada Respiratorias/veterinaria , Tomografía Computarizada por Rayos X/instrumentación , Tomografía Computarizada por Rayos X/veterinaria , Animales , Diseño de Equipo , Análisis de Falla de Equipo , Rayos Láser , Ratones , Ratones Endogámicos C57BL , Movimiento (Física) , Dosificación Radioterapéutica , Reproducibilidad de los Resultados , Mecánica Respiratoria , Sensibilidad y EspecificidadRESUMEN
Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.
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Técnicas Biosensibles/métodos , Neoplasias de la Mama , Receptores ErbB/metabolismo , Neoplasias Mamarias Experimentales , Proteínas de Neoplasias/metabolismo , Transfección/métodos , Microambiente Tumoral , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/genética , Femenino , Fluorescencia , Humanos , Liposomas , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Proteínas de Neoplasias/genética , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismoRESUMEN
A "broadbeam" facility is demonstrated for the vertical microbeam at Surrey's Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651-662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in "broadbeam" mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.
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Radiobiología/métodos , Animales , Línea Celular , Cricetinae , Cricetulus , Relación Dosis-Respuesta en la Radiación , Transferencia Lineal de Energía , Método de Montecarlo , Radiobiología/instrumentaciónRESUMEN
We describe a microscopy design methodology and details of microscopes built to this 'open' design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam 'end-stations' at Oxford and Surrey Universities, showing the versatility and extendibility of this approach.
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Biología/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Automatización de Laboratorios/métodosRESUMEN
OBJECTIVE: To reinvestigate ultra-high dose rate radiation (UHDRR) radiobiology and consider potential implications for hadrontherapy. METHODS: A literature search of cellular UHDRR exposures was performed. Standard oxygen diffusion equations were used to estimate the time taken to replace UHDRR-related oxygen depletion. Dose rates from conventional and novel methods of hadrontherapy accelerators were considered, including spot scanning beam delivery, which intensifies dose rate. RESULTS: The literature findings were that, for X-ray and electron dose rates of around 10(9) Gy s(-1), 5-10 Gy depletes cellular oxygen, significantly changing the radiosensitivity of cells already in low oxygen tension (around 3 mmHg or 0.4 kPa). The time taken to reverse the oxygen depletion of such cells is estimated to be over 20-30 s at distances of over 100 µm from a tumour blood vessel. In this time window, tumours have a higher hypoxic fraction (capable of reducing tumour control), so the next application of radiation within the same fraction should be at a time that exceeds these estimates in the case of scanned beams or with ultra-fast laser-generated particles. CONCLUSION: This study has potential implications for particle therapy, including laser-generated particles, where dose rate is greatly increased. Conventional accelerators probably do not achieve the critical UHDRR conditions. However, specific UHDRR oxygen depletion experiments using proton and ion beams are indicated.
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Iones/uso terapéutico , Neoplasias/radioterapia , Terapia de Protones , Radioterapia de Alta Energía/métodos , Hipoxia de la Célula/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Humanos , Oxígeno/efectos de la radiación , Aceleradores de Partículas , Tolerancia a Radiación , Dosificación Radioterapéutica , Células Tumorales CultivadasRESUMEN
This paper gives an overview of research and patents concerning the use of natural zeolites in water-treatment systems in the last ten years. Furthermore, nanocomposite materials made of natural zeolites and organic and polymeric materials are also mentioned as an effective solution in water treatment. An additional emphasis is put on a variety of possibilities for further application of natural zeolite materials for environment protection and preservation.
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Purificación del Agua/métodos , Zeolitas/química , Adsorción , Nanocompuestos/química , Patentes como Asunto , Polímeros/químicaRESUMEN
We review novel, in vivo and tissue-based imaging technologies that monitor and optimize cancer therapeutics. Recent advances in cancer treatment centre around the development of targeted therapies and personalisation of treatment regimes to individual tumour characteristics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response must address spatial heterogeneity of cancer within the body. A combination of different imaging modalities should be used to relate the effect of the drug to dosing regimen or effective drug concentration at the local site of action. Molecular imaging provides a functional and dynamic read-out of cancer therapeutics, from nanometre to whole body scale. At the whole body scale, an increase in the sensitivity and specificity of the imaging probe is required to localise (micro)metastatic foci and/or residual disease that are currently below the limit of detection. The use of image-guided endoscopic biopsy can produce tumour cells or tissues for nanoscopic analysis in a relatively patient-compliant manner, thereby linking clinical imaging to a more precise assessment of molecular mechanisms. This multimodality imaging approach (in combination with genetics/genomic information) could be used to bridge the gap between our knowledge of mechanisms underlying the processes of metastasis, tumour dormancy and routine clinical practice. Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing an early read-out of response or resistance.
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Biomarcadores de Tumor/análisis , Imagen Molecular/métodos , Proteínas de Neoplasias/análisis , Neoplasias/diagnóstico , Neoplasias/terapia , Humanos , Neoplasias/metabolismo , Integración de SistemasRESUMEN
We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein-protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.
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Polarización de Fluorescencia/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas Biosensibles , Línea Celular , Polarización de Fluorescencia/instrumentación , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Proteínas Fluorescentes Verdes/química , Humanos , Proteínas Luminiscentes/química , Microscopía Fluorescente , Conformación Proteica , Mapeo de Interacción de Proteínas/instrumentación , Mapeo de Interacción de Proteínas/métodos , Sensibilidad y Especificidad , Linfocitos T/química , Factores de Tiempo , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP rac1/química , Proteína Fluorescente RojaRESUMEN
A cellular imaging system, optimized for unstained cells seeded onto a thin substrate, is under development. This system will be a component of the endstation for the microbeam cell-irradiation facility at the University of Surrey. Previous irradiation experiments at the Gray Cancer Institute (GCI) have used Mylar film to support the cells [Folkard, M., Prise, K., Schettino, G., Shao, C., Gilchrist, S., Vojnovic, B., 2005. New insights into the cellular response to radiation using microbeams. Nucl. Instrum. Methods B 231, 189-194]. Although suitable for fluorescence microscopy, the Mylar often creates excessive optical noise when used with non-fluorescent microscopy. A variety of substrates are being investigated to provide appropriate optical clarity, cell adhesion, and radiation attenuation. This paper reports on our investigations to date.
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Células/efectos de la radiación , Diagnóstico por Imagen/métodos , Microscopía/instrumentación , Adhesión Celular , Técnicas Citológicas , Células HeLa , HumanosRESUMEN
A large group of tubulin-binding microtubule-depolymerizing agents act as tumour vascular disrupting agents (VDAs). Several members of this group are now in clinical trials in combination with conventional anticancer drugs and radiotherapy. Here we briefly update on the development of tubulin-binding combretastatins as VDAs, summarize what is known of their mechanisms of action and address issues relating to treatment resistance, using disodium combretastatin A-4 3-O-phosphate (CA-4-P) as an example. Characteristically, VDAs cause a rapid shutdown of blood flow to tumour tissue with much less effect in normal tissues. However, the tumour rim is relatively resistant to treatment. Hypoxia (or hypoxia reoxygenation) induces upregulation of genes associated with angiogenesis and drug resistance. It may be possible to take advantage of treatment-induced hypoxia by combining with drugs that are activated under hypoxic conditions. In summary, VDAs provide a novel approach to cancer treatment, which should effectively complement standard treatments, if treatment resistance is addressed by judicious combination treatment strategies.
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Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias/irrigación sanguínea , Moduladores de Tubulina/uso terapéutico , Animales , Vasos Sanguíneos/efectos de los fármacos , Terapia Combinada , Resistencia a Antineoplásicos , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Ratas , EstilbenosRESUMEN
We report the demonstration of time-correlated single-photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white-light supercontinuum excitation source. By using a 100 fs-pulsed Ti:Sapphire laser with a low-frequency chirp to pump a 30-cm long section of photonic crystal fibre, a ps-pulsed white-light supercontinuum was created. Optical bandpass interference filters were then applied to this broad-bandwidth source to select the 488-nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non-infected material. The white-light generation source provides a flexible method of producing variable-bandwidth visible and ps-pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.
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Caries Dental/patología , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Diente/anatomía & histología , Diseño de Equipo , Humanos , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Óptica y Fotónica/instrumentaciónRESUMEN
A study of the practicality a simple technique for obtaining time-domain information that uses continuous wave detection of fluorescence is presented. We show that this technique has potential for use in assays for which a change in the lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer, for example, and could be particularly important when one is carrying out such measurements in the scaled-down environment of a lab on a chip (biochip). A rate-equation model is presented that allows an objective analysis to be made of the relative importance of the key measurement parameters: optical saturation of the fluorophore and period of the excitation pulse. An experimental demonstration of the technique that uses a cuvette-based analysis of a carbocyanine dye and for which the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode is compared with the model.
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Algoritmos , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Espectrometría de Fluorescencia/métodos , Estroboscopía/métodos , Colorantes Fluorescentes , Semivida , CinéticaRESUMEN
Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug-target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon 'dark' DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug-target interactions with cellular stress responses.
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Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Microscopía Fluorescente/métodos , Modelos Moleculares , Tecnología Farmacéutica , Topotecan/administración & dosificación , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Humanos , Topotecan/metabolismo , Topotecan/farmacocinéticaRESUMEN
Protein-protein interactions and signal transduction pathways have traditionally been analysed using biochemical techniques or standard microscopy. Although invaluable in the delineation of protein hierarchy, these methods do not provide information on the true spatial and temporal nature of complex formation within the intact cell. Recent advances in microscopy have allowed the development of new methods to analyse protein-protein interactions at very high resolution in both fixed and live cells. The present paper provides a brief overview of using fluorescence resonance energy transfer to analyse directly molecular interactions and conformational changes in various proteins involved in the regulation of cell adhesion and motility.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía/métodos , Fosforilación , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.
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Apoptosis/fisiología , Apoptosis/efectos de la radiación , Efecto Espectador/fisiología , Técnicas de Cultivo de Célula/métodos , Transferencia Lineal de Energía/fisiología , Radiometría/métodos , Rayos X , Animales , Efecto Espectador/efectos de la radiación , Células CHO , Técnicas de Cultivo de Célula/instrumentación , Cricetinae , Cricetulus , Relación Dosis-Respuesta en la Radiación , Dosis de RadiaciónRESUMEN
The understanding of tumour angiogenesis is of great importance in cancer research, as is the tumour response to vascular-targeted drugs. This paper presents software aimed at aiding these investigations and other situations where linear or dendritic structures are to be delineated from three-dimensional (3D) data sets. This software application was written to analyse the data from 3D data sets by allowing the manual and semi-automated tracking and delineation of the vascular tree, including the measurement of vessel diameter. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to locate vessel centres and measure diameters automatically. The robustness of this algorithm to image smoothing and noise has been investigated.