Chemical design and biological applications of fluorescence lifetime dyes and sensors

Abstract

Fluorescent probes are proved to be one of the most powerful tools in the past decades, including detecting, quantifying, and visualizing of various molecular interactions and biological processes. However, most of the probes are based on fluorescent intensity as readouts, which are limited for sensitivity and challenge of spectral overlap. Alternatively, fluorescence lifetime (FLT) and fluorescence lifetime imaging microscopy (FLIM) provide time-resolved measurements. FLT is particularly well-suited for monitoring environmental changes because it is independent of concentration but can be sensitive to ion concentration, pH, and temperature etc. It can also be a sensitive tool to trace the interaction between proteins or sensors and analytes due to the shift of FLTs after binding behaviour. Moreover, unlike traditional fluorescence probes, FLT-based probes are better suited for multiplexed imaging due to the addition of the time-resolved dimension. Based on these, we successfully designed new FLIM probes which have good brightness and suitable FLT ranges and tried to answer biological questions using FLT as readouts. In this thesis, we have developed a new chemical library of dyes tailored for FLIM. These dyes either have different core structures or are conjugated with various side chains. We proved that changing of the side chains resulted in different brightness, water solubility and FLT (within the range of 5-25 ns). Additionally, these probes are quite stable and do not undergo photobleaching, which indicates their potential in imaging. Using this innovative FLIM toolbox, we explored FLT variations in different solution conditions, such as the conditions with reactive oxygen and nitrogen species (ROS/RNS). Moreover, we investigated FLT changes associated with DNA binding and DNA oxidative damage in cancer cell lines, providing valuable insights into FLIM probes as sensors for cellular oxidative stress and its broader implications, including their potential role in chronic neuropathic pain, which is associated with DNA oxidative damage. We further enhanced the selectivity of our designed dyes by conjugating them with peptides (e.g., antimicrobial peptides (AMPs)) and small inhibitors. The FLIM settings were optimized, and images reflecting FLTs were acquired. Data analysis of FLT was also refined to enhance accuracy and reliability. We have demonstrated the FLIM probes designed are conjugatable with peptides and small drugs, and achieved selectively targeting and obtained different readouts in different microenvironments. We also partially achieved multiplexing with this toolbox, shedding light on future real-time tracking of dynamic activities in cells