RESUMEN
Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure.
Asunto(s)
Apoptosis , Transferencia Resonante de Energía de Fluorescencia , Caspasa 3/genética , Caspasa 3/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/fisiologíaRESUMEN
A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.
Asunto(s)
Imagenología Tridimensional/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Humanos , Imagenología Tridimensional/instrumentación , Células MCF-7 , Técnicas Analíticas Microfluídicas/instrumentación , Microscopía Fluorescente/instrumentación , Esferoides CelularesRESUMEN
A new concept of three-dimensional imaging of tumor cell spheroids by light sheet-based fluorescence microscopy and nanosecond ratio imaging is described. Due to its low light dose and alternative excitation by two laser wavelengths (391 and 470 nm), this method maintains cell viability and permits recording of real-time kinetics. A genetically encoded sensor permits measurement of the redox state of glutathione and visualization of the impact of oxygen radicals. The pharmaceutically relevant system is tested upon addition of an oxidizing agent (H2O2), as well as upon addition of the apoptosis-inducing agent staurosporine.
Asunto(s)
Técnicas de Cultivo de Célula , Microscopía Fluorescente/métodos , Esferoides Celulares , Células Tumorales Cultivadas , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Humanos , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción/efectos de los fármacos , Esferoides Celulares/química , Esferoides Celulares/citología , Estaurosporina/farmacología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citologíaRESUMEN
A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 µm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.
