WUR needs a contract for the delivery of a microscope system, that is capable of 3D imaging of molecular and cellular functioning in living tissues, with an optical resolution below the diffraction limit. The general aim of node 2 of the NL-bioimaging consortium is to apply 3D functional imaging in complex living-cellular systems. 3D imaging of multicellular organisms like organoids or plants allows to obtain the landscape of molecular events and to reveal the mode of action of proteins in their natural habitat. Specifically at WUR, the focus of the 3D functional imaging node will be on ‘high resolution’, ‘endogenous expression’ and ‘FLIM/FCS’. The main requirement for the new system is that specimen can be imaged with a resolution beyond the diffraction limit (super-resolution microscopy or optical nanoscopy). The system should be able to image fluorescent proteins (FPs) expressed at endogenous levels in living plant tissues. Furthermore, the system should have functional imaging tools, such as fluorescence lifetime imaging microscopy (FLIM). Moreover, the conditions for super-resolution should not interfere with (multi-)cellular functioning. The main challenge for the system is to suppress autofluorescence in these samples. Fluorescence background from e.g. chloroplasts in living plant cells or tissues is typically much stronger than the signals from the fluorescent proteins of interest. The detection part of the microscope should therefore be able to deal with a high number of photons, and it should be able to distinguish weak signals of interest from strong background fluorescence. Proven ways how this can be achieved are tuneability of excitation and emission wavelength (white light laser, tuneable filters, spectral unmixing) and/or time gating with photon counting detectors. The second challenge is to go from 2D to 3D functional imaging. This means that more spatial information must be obtained within the same – sample and experiment dependent – time window than before. We therefore seek a 3D FLIM system that is fast allowing real time 3D visualization of the lifetime information (or simply lifetime contrast). This is prioritized over high time resolution FLIM that requires post processing of the data. When experiments require more detailed information, FLIM and fluorescence correlation spectroscopy (FCS) with high temporal resolution and accuracy can be performed (in 2D) on the currently available laser scanning microscopes within the MSC.