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 The main
goal of the Probe Development subproject is the development of novel fluorescent
probes and sensors with a large range of wavelengths. Particular aims are:
- Generation and optimization of far-red fluorescent proteins (FP)
suitable for imaging in tissue.
- Generation and optimization of photoactivated Kindling Fluorescent
Proteins (KFP).
- Generation of a novel set of red-fluorescent dyes with excitation maxima
at 543 or 594 nm and emission maximum >650 nm. Incorporation of these dyes
in sensors and modification of the sensors to become membrane-permeant,
bioactivatable derivatives.
- Development of new chemical multiphoton cages.
- Development of advanced FRET pairs based on novel FPs, KFPs and chemical
fluorophores.
- Development of new or improved biosensors based on novel FPs, red
photoactivated KFPs, modified fluorescent dyes and multiphoton cages.
Existing and novel probes and biosensors will be
exploited in specifically engineered biotechnological systems to assess their
suitability for usage in the imaging devices described below and in parallel
will be used to improve specific features such as resolution, sensitivity, and
multispectral capability.
In particular:
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Well established and improved systems will be
used to express efficiently fluorescent proteins in cells and transgenic
animals. Transgenic mouse lines with genetically encoded tags will be
generated and tested for their applicability to monitor cell trafficking,
intracellular protein trafficking and interactions within living cells in
real time.
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A collection of novel sensors will be
generated for monitoring in vivo biological processes such as:
Phospholipases A2 and C activities, Kinase/phosphatase activity, Lipid
signaling activity, Chemoattractant receptor activation and lymphocyte
trafficking and finally T cell signaling.
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