Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides

Antisense oligonucleotides (AON) have been intensively studied as tools in molecular cell biology and as novel therapeutics in various diseases over the past two decades. Especially cellular uptake and endosomal release of AONs are topics of interest, as these are crucial steps in reaching the subcellular AON target sites and achieving biological activity. We used cell-penetrating peptides (CPPs) to enhance uptake and endosomal release of AONs, and monitored these two processes and the subsequent fate of the AONs by advanced fluorescence microscopy in living cells. In this chapter, we discuss the use of automated time-lapse confocal laser scanning microscopy (CLSM) to follow AON uptake and trafficking in time, fluorescence lifetime imaging microscopy (FLIM) to distinguish between free and AON-bound fluorophore, and fluorescence correlation spectroscopy (FCS) to measure subcellular AON concentrations and molecular associations. Additionally, we expand on the analysis of these microscopy data.