Live cell spinning disk confocal microscope with single molecule localization module

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Fluorescence light microscopy allows to follow subcellular dynamics of protein complexes or – if the signal is bright enough - even of single proteins. Due to the diffraction limit of light, it is not possible to resolve signals that are spaced closer than approximately half the wavelength of the used light. Confocal microscopy helps to improve contrast and eliminate out of focus signal but does not overcome the diffraction limit. In this proposal, we want to use spinning disk microscopy as a highly parallelized and therefore fast way of confocal imaging combined with a super-resolution modality, to achieve the best confocal contrast inside cells with ultimate sensitivity and high temporal resolution in 3D as well as near molecular resolution. This will allow answering questions on localization of proteins and RNAs to organelles or other specific sites of the cell identified by particular markers. It will also allow analyzing the interaction between glioblastoma cells and neurons forming an excitatory synapse. We cannot predict exactly when formation occurs, i.e., we need to capture with a high frame rate volumetric data over an extended period of time. We therefore ask for a spinning disk confocal system which allows us to collect many frames without inflicting phototoxicity or other disturbances. In addition, and to overcome the diffraction-based limitation in resolution, we ask for a modality for single molecule localization microscopy (SMLM) to perform super-resolution microscopy on the same specimens. This modality will allow for up to 3 channels based on the combination of STORM, DNA-paint or PALM and results in an xy resolution of 20 nm. 3D information will be obtained by a spherical lens in the emission light path and several micrometer will be covered by merging the super-resolution stacks acquired at different z-positions. While highest contrast is achieved using TIRF illumination, we want to also several micrometer into the cell, and therefore need a way to automatically decrease the incidence angle of the laser to allow illumination beyond the TIRF field. Importantly, all components of the multimodal instrument need to be motorized and controlled by software. This allows to save user specific configurations, a prerequisite for placing the system into a multi-user imaging facility, as proposed here. In summary, the requested multimodal microscope setup will advance our experimental abilities in two main directions. 1) Sensitive and fast live imaging of subcellular structures in 3D with spinning disk confocal microscopy. 2) Subsequent study of localization of single proteins and protein complexes in fixed cells at 20 nm. Importantly, by using the same platform, we can find xy points in the sample again and correlate live imaging with super-resolution microscopy data.