A Novel Fluorescent Dye Extracted from Buddleja officinalis for Labeling Mitochondria after Fixation

Abstract

Mitochondria are versatile organelles and function by communicating with cellular ecosystems. The fluorescent colocalization analysis after fixation is a highly intuitive method to understand the role of mitochondria. However, there are few fluorescent dyes available for mitochondrial staining after fixation. In this study, a novel fluorescent dye (BO-dye), extracted from Buddleja officinalis, was applied for mitochondrial staining in fixed immortalized human oral keratinocytes. The BO-dye (excitation: 414 nm, emission: 677 nm) is a small fluorescent molecular dye, which can cross the cytomembrane without permeabilization. We assume that the BO-dye could aggregate and bind to the mitochondria stably. BO-dye exhibited a mega-Stokes shift (>250 nm), which is an important feature that could reduce self-quenching and enhance the signal-to-noise ratio. Analysis of photophysical properties revealed that the BO-dye is temperature and pH insensitive, and it exhibits superior photostability. These results indicate that BO-dye can be considered an alternative fluorescent dye for labeling mitochondria after fixation.

Expansion microscopy for super-resolution imaging of collagen-abundant tissues

“…In ColExM, we employed a streptavidin-based technique to enhance fluorescent signals, thereby mitigating the reduction in fluorescent signals caused by increased molecular distances during the expansion process. Due to its compatibility with immunostaining, ColExM is also possibly compatible with commercially available organelle-staining dyes, such as Lysotracker 44 and MitoView TM Green 45 . For example, the incorporation of MitoView TM Green into ColExM of clinical specimens may offer the potential to explore the mechanisms of mitochondrial dysfunction and mitophagy in the pathogenesis of Fuchs endothelial corneal dystrophy in future studies.…”