Quantitative 3D and 4D cell analysis in living organisms -
novel instrumentation, computational tools, proof of concept applications

part of the BMBF-supported programme: "New Methods in Systems Biology" (SYSTEC)

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Within this project we apply quantitative imaging to analyze subcellular distribution and co-localization of proteins in populations of living cells. The main imaging techniques are fluorescence resonance energy transfer (FRET) microscopy measurements to define the spatial relationships between proteins, or fluorescence lifetime imaging microscopy (FLIM) for intensity-based measurements to detect localized protein interactions with spatial resolution. This includes the development and exploration of novel fluorescent sensors as an alternative to GFPs to reduce bleaching and phototoxic effects to living cells, development of quantitative optical measurements in living cells that enable monitoring of dynamic molecule interactions using FRET or FRET-based sensors and determining the kinetic behaviour in vivo. Database systems need to be developed for standardized, certified recording, documentation and validation of optical data and exchange between different users or laboratories.

Optical methods will be of central relevance for qualitative and quantitative analysis of cells and tissues as they allow non-destructive observation of cellular dynamics at high spatial (3D) and temporal resolution (3+1D) in live cells. However, currently used technologies still have major shortcomings such as a lack of rigorous standardisation and quantisation. They would greatly benefit from novel methods and algorithms for data acquisition, analysis, modelling and evaluation. Thus techniques and appliances for three-to-four-dimensional, multi-parameter imaging from single cells to multi-cellular systems such as organs need to be developed, and instrumentation for the robust, standardized quantitation of cellular pathways need to become available for high-throughput analysis.

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