Lifetime changes Development of genetically encoded biosensors for quantitative imaging

To understand cellular behavior, biologists need tools to visualize intracellular changes in real time. One option are fluorescent biosensors, that report with a change in fluorescence in the presence or absence of a specific analyte (ions, sugars, small molecules). Combining them with microscopy allows researchers to visualize intracellular processes with high spatiotemporal resolution. Genetically encoded fluorescent biosensors are developed using a fluorescent protein (FP), fused to a protein domain that binds an analyte. Many biosensors have been developed over the years for a wide range of analytes in colors spanning the full visual spectrum, and they can be expressed in virtually any organism by introducing the genetic code.
Most biosensors respond with a change in the fluorescence intensity of the sensor upon binding of the analyte, revealing if there is a change in the level of the analyte. Quantification, however, is challenging, because the intensity is influenced by other factors such as: expression level of the sensor, cell movement and thickness, microscope hard- and software. This thesis focusses on development of biosensors for quantitative imaging, by creating sensors that show a change in the fluorescence lifetime upon binding of an analyte. The fluorescence lifetime is the average time between excitation and emission of a fluorescent molecule and is independent of the factors mentioned above, allowing for quantitative imaging. Calcium was chosen as the analyte of interest, due to its central role in many intracellular processes. Sensors were created in different colors, thoroughly characterized, and applied in various mammalian cell types.