RESUMO
Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
Assuntos
Imageamento Tridimensional , Microscopia Intravital , Movimento (Física) , Algoritmos , Animais , Técnicas Biossensoriais , Adesão Celular , Simulação por Computador , Transferência Ressonante de Energia de Fluorescência , Guanosina Trifosfato/metabolismo , Humanos , Intestinos/fisiologia , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Metástase Neoplásica , Neuropeptídeos/metabolismo , Neoplasias Pancreáticas/patologia , Pele/anatomia & histologia , Software , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Numerous large scale genomics studies have demonstrated that cancer is a molecularly heterogeneous disease, characterized by acquired changes in the structure and DNA sequence of tumor genomes. More recently, the role of the equally complex tumor microenvironment in driving the aggressiveness of this disease is increasingly being realized. Tumor cells are surrounded by activated stroma, creating a dynamic environment that promotes cancer development, metastasis and chemoresistance. The Rho family of small GTPases plays an essential role in the regulation of cell shape, cytokinesis, cell adhesion, and cell motility. Importantly, these processes need to be considered in the context of a complex 3-dimensional (3D) environment, with reciprocal feedback and cross-talk taking place between the tumor cells and host environment. Here we discuss the role of molecular networks involving Rho GTPases in cancer, and the therapeutic implications of inhibiting Rho signaling in both cancer cells and the emerging concept of targeting the surrounding stroma.