Two-photon-confocal microscope incl. FLIM detectors

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While the biochemical and genetic impact on proliferation and differentiation has been addressed down to the molecular detail, the role of forces in cell fate decisions is just started to be addressed. It is known that the elasticity of the substrate crucially impacts on the differentiation route of cultured cells. The direct measurement of the decisive elements in the organismal context was limited by the absence of molecular tools and microscopes with the necessary penetration depth. The progress in Crispr/Cas9 based genome editing, the establishment of genetically encoded force sensors, combined with the progress in multi-photon confocal microscopy and here in particular the fluorescence life time imaging (FLIM) has opened the door for in vivo analyses in the developing regenerating and aging organism. The establishment and maintenance of the retinal stem cell niche (CMZ) in fish, a focus of our research for years, appears to be crucially dependent on forces. The CMZ is located at the interface between the neural retina and the retinal pigmented epithelium and resides in a prominent “bending zone”. With the instrument applied for we are (among other points) aiming to address the role of physical forces acting on the stem and progenitor cells in the organismal context, in vivo. This will be facilitated by the use of genetically encoded force sensors (e.g. Talin-FRET sensors, vinculin-FRET sensors, Abl-kinase FRET-sensors), integrated into the genomic loci of the model by homology directed repair. The instrument applied for will allow to measure acting forces by the force dependent loss of the FRET signal. Clonal analysis will facilitate the correlation of forces with the developmental fate of stem and progenitor cells. Retinal organoids established in the lab will eventually allow directly testing the hypotheses substantiated by the in vivo analyses. This will be achieved by direct and directed force application to the organoids to address their action in an out of context setting. The instrument applied for will be crucial to perform those analyses and close the loop of arguments. The instrument will be a mandatory prerequisite for the scientific progress of four (junior) groups at COS. The Lemke lab specializes in high-resolution imaging of cytoskeletal and motor proteins in several different insect species, with strong auto-fluorescence. FLIM allows the discrimination of such auto-fluorescence from the actual signal, enabling cross-species comparisons otherwise not possible. The Centanin, Bageritz, and Weinhardt labs all depend on fast multicolour acquisition of larger numbers of fluorophores with overlapping emission spectra. FLIM microscopy facilitates larger numbers of co-labelled probes, enabling precise hypothesis testing as well as reducing the total number of experiments and animals required. The access to the applied instrument will ensure that all user groups can pursue projects at a level that is internationally competitive.