Contrast enhancement of fluorescent biosensors through non-linear signal amplification of ground state dimers

Objectives

Fluorescence-based biosensors are indispensable for spatially resolved microscopy applications in vivo and in vitro. However, a general limitation of such sensors is the linear signal increase with their abundance, leaving the quality of the signal to background ratio only to the specificity of the sensor itself. Here, we overcome this general limitation by employing our recently discovered ground- state dimers of BODIPY conjugates (D||), which spontaneously form in the environment to which they localize. These short-lived BODIPY- D|| states can be specifically excited and detected at the single molecule level with red-shifted wavelengths, which dramatically enhances their signal to background ratio compared to detecting the monomeric form. We aim to develop this approach in vitro and in live mammalian cells for improved biosensing applications and to expand the toolbox to the myriad of existing BODIPY conjugates.

Expected Outcome

We expect to develop BODIPY-D|| imaging into a well characterized biosensing modality. By optimizing dye concentrations and measuring environmental influences such as temperature and pH the best experimental conditions will be explored. The application to fatty acid trafficking within and between mammalian cells will yield new insights into fat metabolism. Finally we expect to expand BODIPY-D|| imaging to ten other BODIPY based biosensors e.g. for detecting lipid peroxidation, sphingolipid transport and labeling specific organelles such as the endoplasmic reticulum, mitochondria and the golgi.