RESUMEN
In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.
Asunto(s)
Anticuerpos/análisis , Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Microscopía Fluorescente/métodos , Anticuerpos/química , Línea Celular Tumoral , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Leucocitos/citología , Leucocitos/efectos de la radiación , Fotoblanqueo/efectos de la radiación , Ficoeritrina/metabolismo , Coloración y Etiquetado , Rayos UltravioletaRESUMEN
OBJECTIVE: The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. EXPERIMENTAL DESIGN: The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [n = 8], and footpad injection [n = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2-fluorescence and flat-panel volumetric computed tomography scan. In a further series of animals (n = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable-magnification small-animal imaging system was used. RESULTS: Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat-panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat-panel volumetric computed tomography scan imaging data was observed. Single-cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. CONCLUSION: We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells.
Asunto(s)
Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Rabdomiosarcoma/patología , Rabdomiosarcoma/secundario , Pantallas Intensificadoras de Rayos X , Animales , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Interpretación de Imagen Asistida por Computador/instrumentación , Imagenología Tridimensional/instrumentación , Proteínas Luminiscentes/química , Ratones , Ratones Desnudos , Microscopía Fluorescente , Invasividad Neoplásica , Metástasis de la Neoplasia , Neovascularización Patológica/patología , Sensibilidad y Especificidad , Células Tumorales Cultivadas , Proteína Fluorescente RojaRESUMEN
The multiparametric molecular cell and tissue analysis in vitro and in vivo is characterized by rapid progress in the field of image generation technologies, sensor biotechnology, and computational modeling. Fascinating new potentials in unraveling the detailed functions of single cells, organs, and whole organisms are presently emerging and permit the close monitoring i.e. tumor development or basic cell development processes with an unprecedented multiplicity of promising investigative possibilities. To answer basic questions of in vivo tumor development and progression fluorescence based imaging techniques provide new insights into molecular pathways and targets. Genetic reporter systems (eGFP, DsRED) are available and high sensitive detection systems are on hand. These techniques could be used for in vitro assays and quantified e.g. by microscopy and CCD based readouts. The introduction of novel fluorescent dyes emitting in the near infrared range (NIR) combined with the development of sensitive detector systems and monochromatic powerful NIR-lasers for the first time permits the quantification and imaging of fluorescence and/or bioluminescence in deeper tissues. Laser based techniques particularly in the NIR-range (like two-photon microscopy) offer superb signal to noise ratios, and thus the potential to detect molecular targets in vivo. In combination with flat panel volumetric computed tomography (fpVCT), questions dealing e.g. with tumor size, tumor growth, and angiogenesis/vascularization could be answered noninvasively using the same animal. The resolution of down to 150 microm/each direction can be achieved using fpVCT. It is demonstrated by many groups that submillimeter resolutions can be achieved in small animal imaging at high sensitivity and molecular specificity. Since the resolution in preclinical small animal imaging is down to approximately 10 microm by the use of microCT and to subcellular resolutions using ( approximately 1 microm) microscope based systems, the advances of different techniques can now be combined to "multimodal" preclinical imaging and the possibilities for in vivo intravital cytometry now become within one's reach.