PROBES FOR CLEM

Innovation and application of genetically-encoded probes for correlated live-cell imaging and electron microscopy

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. The possibilities are endless: the probes can be used to study proteins in live or fixed specimens; in vitro or in vivo; to study localization or activity, or modify function; and by light and electron microcopy. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. However, the fluorescent toolbox has become very complex and each tool has specific requirements and pros and cons for different applications. The aim of this project is to develop, validate and implement a probe for combined fluorescence live-cell imaging and electron microscopy of proteins of interest. The combinatorial probe will be based on a genetically-encoded tag consisting of a fluorescent protein (highly suitable for live cell imaging) and horseradish peroxidase to visualize proteins by electron microscopy at high resolution with high quality preservation of the ultrastructure. In addition, we will explore the general application of a recently introduced manner of increasing resolution using light microscopy (photoactivatable localization microscopy; PALM). While the focus of this project proposal is to create probes for general application in cell biology, initially these probes will be implemented in my studies that aim to resolve the role of cell-cell contacts in regulating beta cell proliferation.'

Introduzione (Teaser)

Microscope technology has come a long way, allowing researchers to characterise and localise protein cell behaviour in an unprecedented manner.

Descrizione progetto (Article)

Medicine relies heavily on precise imaging systems to observe protein cells, map their behaviour, diagnose disease and even postulate treatments. In general, electron microscopes that produce an electronically magnified image are used to observe these kinds of cells.

Recent advances in organic chemistry, molecular biology and materials science are enabling improvements in microscope technology. An enterprising European project is creating several new classes of fluorescent probes that can be used for imaging in cell biology. The project, fully funded by the EU, is entitled 'Innovation and application of genetically encoded probes for correlated live-cell imaging and electron microscopy?. It is creating endless possibilities as the probes can be used to study proteins in live or fixed specimens, as well as localise activity of the proteins or even modify their function. The ultimate aim of this project is to develop a probe with two combined technologies, known as fluorescence live-cell imaging and electron microscopy (Clem), which simultaneously target specific proteins.

Increasing the resolution of the imaging is also being investigated, as is light-based microscopy, formally known as photoactivatable localisation microscopy (Palm). The project team has made great strides in its research, successfully introducing molecular biology and cell culture in a traditional electron microscopy lab environment. The combined technologies microscope was developed and tested, resulting in the 'first genuine genetically-encoded tag that allows for live-cell imaging and correlated electron microscopy'. Overall microscopic imaging was improved, and observation of larger areas with the new microscope accomplished. The technology now enables imaging at very fine detail or 'ultrastructural' level, with highly specific targeting. Moreover, since the probe is genuinely 'genetically encoded' and easy to use compared to current fluorescent protein technology, it can be made readily available to the entire cell biology research community.