1. Bioimaging Facility - Widefield + confocal microscopes, workstations for image analysis - basic + advanced usage - training to achieve independent usage Standard applications: - Transfection checks - Cell count - Quality control - Multichannel fluorescence for localisation (2D) - 3D localisation + visualisation - Illustration Advanced (collaborative): Live cell imaging, colocalisation, quantification, FRET, FRAP, spectral imaging, biosensors, rapid imaging + tracking, low phototoxicity imaging

2. Colocalisation Background - Indicator of interaction between two subcellular structures - Common first step in routine biomedical laboratories - Often qualitatively assessed (red + green = yellow) - easier + more robust than FRET Research questions Does HLA-G colocalise with other surface receptors?  Method for quantified colocalisation of punctate structures with 30nm accuracy (Obara et al., HCB, 2012) Do such associations change in different conditions?  Yup (Jabeen et al., Hum Reprod, 2013)

4. Quantified phenotyping Background Cellular model for Autosomal Dominant Retinitis Pigmentosa (ADRP): HEK293S cells expressing WT Rhodopsin-GFP or rhodopsin mutants Misfolded rhodopsin leads to protein aggregates inside cells Amount of signal in plasma membrane corresponds to folding (Goncalves et al., MiE, 2012) Use of molecular chaperones may rescue misfolding mutants WT P23H P23H rescued

5. Conclusions Work in progress (FOM 2013) Rule out variability at sample level Variability in transiently transfected (then fixed) cells very high  stable cell lines, inducible, identical image acquisition parameters, high number of datasets  Image library to test algorithms  Quality of sample + acquired images crucial Visual vs automated analysis  Algorithms must be carefully evaluated + adapted if necessary  Human eye good for patterns computer analysis for gradients  Semi-quantitative assessment valid first approach  Multi-parametric analysis; dimensions counter variability  Live cell imaging adds time  Potential down-regulation of calnexin in P23H mutant