Cell Imaging Platforms (CIPs) are research infrastructures offering support to a number of scientific projects including the choice of adapted fluorescent probes for live cell imaging. What to detect in what type of sample and for how long is a major issue with fluorescent probes and, for this, the “hat-trick” “Probe–Sample–Instrument” (PSI) has to be considered. We propose here to deal with key points usually discussed in CIPs including the properties of fluorescent organic probes, the modality of cell labeling, and the best equipment to obtain appropriate spectral, spatial, and temporal resolution. New strategies in organic synthesis and click chemistry for accessing probes with enhanced photophysical characteristics and targeting abilities will also be addressed. Finally, methods for image processing will be described to optimize exploitation of fluorescence signals.
