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Volker Hilsenstein, Sabine Reither, Beate Neumann, Christian Tischer, Rainer Pepperkok (EMBL Advanced Light Microscopy Facility, Heidelberg, Germany)
In fluorescence microscopy, the number of proteins that can be imaged in the same sample is limited by the spectral overlap of dyes. With a typical fluorescence microscope up to four fluorescent markers can be imaged routinely. One can push the number of proteins that can be imaged in the same cell significantly by using multiple cycles of labelling, imaging and bleaching with different primary labelled antibodies. We have fitted a commercial confocal microscope with a pipetting device to build an automated system for such multilabel imaging. We demonstrate visualization of up to 14 different markers in the same cell at confocal resolution. Localisation and morphological information of many proteins gives insight into their interaction, but the multitude of channels also present a challenge for interacting with the data. We propose a multi-channel viewer software which allows exploration of different combinations of proteins using separate layers. While this viewer software is a first step, we seek feedback from the visualisation community on how to interact with such multi-channel data.