RESUMEN
Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.
Asunto(s)
Receptores ErbB/química , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Fotoblanqueo , Animales , Cricetinae , Femenino , Humanos , Sustancias MacromolecularesRESUMEN
There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5-50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm.
Asunto(s)
Factor de Crecimiento Epidérmico/química , Receptores ErbB/química , Sustancias Macromoleculares/química , Colorantes Fluorescentes/química , Humanos , Multimerización de ProteínaRESUMEN
Multiphoton microscopy is widely employed in the life sciences using extrinsic fluorescence of low- and high-molecular weight labels with excitation and emission spectra in the visible and near infrared regions. For imaging of intrinsic and extrinsic fluorophores with excitation spectra in the ultraviolet region, multiphoton excitation with one- or two-colour lasers avoids the need for ultraviolet-transmitting excitation optics and has advantages in terms of optical penetration in the sample and reduced phototoxicity. Excitation and detection of ultraviolet emission around 300 nm and below in a typical inverted confocal microscope is more difficult and requires the use of expensive quartz optics including the objective. In this technical note we describe the adaptation of a commercial confocal microscope (Nikon, Japan E-C1 or E-C2) for versatile use with Ti-sapphire and OPO laser sources and the addition of a second detection channel that enables detection of ultraviolet fluorescence and increases detection sensitivity in a typical fluorescence lifetime imaging microscopy experiment. Results from some experiments with this setup illustrate the resulting capabilities.
Asunto(s)
Microscopía Confocal/instrumentación , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Apoptosis , Diseño de Equipo , Colorantes Fluorescentes , Células Tumorales CultivadasRESUMEN
Photodynamic therapy (PDT) is an alternative cancer treatment to conventional surgery, radiotherapy and chemotherapy. It is based on activating a drug with light that triggers the generation of cytotoxic species that promote tumour cell killing. At present, PDT is mainly used in the treatment of wet age-related macular degeneration, for precancerous conditions of the skin (e.g. actinic keratosis) and in the palliative care of advanced cancers, for instance of the bladder or the oesophagus. PDT is still not used as a first line cancer treatment, which is surprising given the first clinical trials by Dougherty's group dating back to the 1970's. PDT has significant advantages over surgery or radiation therapy for low lying tumours due to better cosmetic outcome and localised treatment for the patients. However, despite these advantages and significant developments in optical technology that has enabled light penetration to deeper lying tumours, in excess of 5 cm, a lack of phase III clinical trials has slowed down the uptake of PDT by the healthcare sector as a frontline treatment in cancer. However research continues to demonstrate the potential benefits of PDT and the need to stimulate funding and uptake of clinical studies using next generation photosensitizers offering advanced targeted delivery, improved photodynamic dose combined with modern light delivery technologies. This review surveys the available PDT treatments and emerging novel developments in the field with a particular focus on two-photon techniques that are anticipated to improve the effectiveness of PDT in tissues at depth and on next generation drugs that work without the need of the presence of oxygen for photosensitization making them effective where hypoxia has taken hold.
Asunto(s)
Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Sistemas de Liberación de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Humanos , Degeneración Macular/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Fotoquimioterapia/instrumentación , Fotones , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/química , Lesiones Precancerosas/tratamiento farmacológicoRESUMEN
The photoisomerization of relatively nontoxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one- and two-photon excitations at 340 and 625 nm, respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygen-independent and active in hypoxic environments such as in tumors. The use of red or near-infrared (NIR) light for two-photon excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes nondiscriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of two-photon red or NIR excitation for deeper tissue penetration.
Asunto(s)
Antineoplásicos/química , Bibencilos/química , Fototerapia/métodos , Animales , Anexina A5/química , Células CHO , Muerte Celular , Supervivencia Celular , Cricetinae , Cricetulus , Ensayos de Selección de Medicamentos Antitumorales , Hidrazinas/química , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Permeabilidad , Fotones , Fármacos Fotosensibilizantes/química , Propidio/química , Isoformas de Proteínas , Espectroscopía Infrarroja CortaRESUMEN
The uptake of E -combretastatins, potential prodrugs of the anticancer Z -isomers, into multicellular spheroids has been imaged by intrinsic fluorescence in three dimensions using two-photon excited fluorescence lifetime imaging with 625-nm ultrafast femtosecond laser pulses. Uptake is initially observed at the spheroid periphery but extends to the spheroid core within 30 min. Using agarose gels as a three-dimensional model, the conversion of Z(trans)âE(cis) via two-photon photoisomerization is demonstrated and the location of this photochemical process may be precisely selected within the micron scale in all three dimensions at depths up to almost 2 mm. We discuss these results for enhanced tissue penetration at longer near-infrared wavelengths for cancer therapy and up to three-photon excitation and imaging using 930-nm laser pulses with suitable combretastatin analogs.
Asunto(s)
Antineoplásicos/farmacocinética , Bibencilos/farmacocinética , Imagenología Tridimensional/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Esferoides Celulares/metabolismo , Antineoplásicos/química , Bibencilos/química , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Fotones , Células Tumorales Cultivadas/metabolismoRESUMEN
Dimerisation, oligomerisation, and clustering of receptor molecules are important for control of the signalling process. There has been a lack of suitable methods for the study and quantification of these processes in cells. Here we describe a protocol for a method that we have named "fluorophore localisation imaging with photobleaching" (FLImP), which uses single molecule localisation and single-step photobleaching to determine the separation of two fluorophores with a resolution of 7 nm or better. We describe the procedures required for the collection of FLImP data, and point out some of the pitfalls that must be avoided for the collection of high resolution data. We also present recent data obtained using FLImP, showing that the intracellular domain of the Epidermal Growth Factor Receptor is not required in the basal state for the receptor to form ordered inactive oligomers in the plasma membrane.
Asunto(s)
Receptores ErbB/química , Imagen Óptica/métodos , Multimerización de Proteína , Animales , Receptores ErbB/metabolismo , Humanos , Espacio Intracelular/metabolismo , Fotoblanqueo , Estructura Terciaria de ProteínaRESUMEN
The initial events after photoexcitation and photoionization of α-tocopherol (vitamin E) and the analogue Trolox C have been studied by femtosecond stimulated Raman spectroscopy, transient absorption spectroscopy and time-resolved infrared spectroscopy. Using these techniques it was possible to follow the formation and decay of the excited state, neutral and radical cation radicals and the hydrated electron that are produced under the various conditions examined. α-Tocopherol and Trolox C in methanol solution appear to undergo efficient homolytic dissociation of the phenolic -OH bond to directly produce the tocopheroxyl radical. In contrast, Trolox C photochemistry in neutral aqueous solutions involves intermediate formation of a radical cation and the hydrated electron which undergo geminate recombination within 100 ps in competition with deprotonation of the radical cation. The results are discussed in relation to recently proposed mechanisms for the reaction of α-tocopherol with peroxyl radicals, which represents the best understood biological activity of this vitamin.
Asunto(s)
Cromanos/química , Procesos Fotoquímicos , Análisis Espectral , Vibración , alfa-Tocoferol/análogos & derivados , Absorción Fisicoquímica , Antioxidantes/química , Radicales Libres/química , Rayos Infrarrojos , Modelos Moleculares , Conformación Molecular , Teoría Cuántica , Factores de TiempoRESUMEN
To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of E-combretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to two orders of magnitude higher than that of the drug in the surrounding medium.