Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

Fast 4D Microscopy.

Many cellular processes involve fast movements of weakly labeled cellular structures in all directions, which should be recorded in 3D time-lapse microscopy (4D microscopy). This chapter introduces fast 4D imaging, which is used for sampling the cell's volume by collecting focal planes in time-lapse mode as rapidly as possible, without perturbing the sample by strong illumination. The final images should contain sufficient contrast allowing for the isolation of structures of interest by segmentation and the analysis of their intracellular movements by tracking. Because they are the most sensitive, systems using wide-field microscopy and deconvolution techniques are discussed in greater depth. We discuss important points to consider, including system components and multifunctionality, spatial resolution and sampling conditions, and mechanical and optical stability and how to test for it. We consider image formation using high numerical aperture optics and discuss the influence of optical blur and noise on image formation of living cells. Spherical aberrations, their consequences for axial image quality, and their impact on the success of deconvolution of low intensity image stacks are explained in detail. Simple protocols for acquiring and treating point spread functions (PSFs) and live cells are provided. A compromise for counteracting spherical aberration involving the use of a kit of immersion oils for PSF and cell acquisition is illustrated. Recommendations for evaluating acquisition conditions and deconvolution parameters are given. Finally, we discuss future developments based on the use of adaptive optics which will push back many of today's limits.

Enhanced fluorescence cell imaging with metal-coated slides.

Fluorescence labeling is the prevailing imaging technique in cell biology research. When they involve statistical investigations on a large number of cells, experimental studies require both low magnification to get a reliable statistical population and high contrast to achieve accurate diagnosis on the nature of the cells' perturbation. Because microscope objectives of low magnification generally yield low collection efficiency, such studies are limited by the fluorescence signal weakness. To overcome this technological bottleneck, we proposed a new method based on metal-coated substrates that enhance the fluorescence process and improve collection efficiency in epifluorescence observation and that can be directly used with a common microscope setup. We developed a model based on the dipole approximation with the aim of simulating the optical behavior of a fluorophore on such a substrate and revealing the different mechanisms responsible for fluorescence enhancement. The presence of a reflective surface modifies both excitation and emission processes and additionally reshapes fluorescence emission lobes. From both theoretical and experimental results, we found the fluorescence signal emitted by a molecular cyanine 3 dye layer to be amplified by a factor approximately 30 when fluorophores are separated by a proper distance from the substrate. We then adapted our model to the case of homogeneously stained micrometer-sized objects and demonstrated mean signal amplification by a factor approximately 4. Finally, we applied our method to fluorescence imaging of dog kidney cells and verified experimentally the simulated results.

A guided tour into subcellular colocalization analysis in light microscopy.

It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.