A gentle palette of plasma membrane dyes

Abstract

Plasma membrane (PM) stains are important organelle markers for monitoring membrane morphology and dynamics. The state-of-the-art PM stains are bright, specific, fluorogenic, and compatible with superresolution imaging. However, when recording membrane dynamics using advanced fluorescence microscopes, PM is prone to photodynamic damage introduced by dyes due to its phospholipid bilayer nature. Here, we introduce PK Mem dyes tailored for time-lapse fluorescence imaging. By integrating triplet-state quenchers into the MemBright dyes featuring cyanine chromophores and amphiphilic zwitterion anchors, PK Mem dyes exhibited a three-fold reduction in phototoxicity and a more than four-fold improvement in photostability in imaging experiments compared to MemBright prototypes. These dyes enable 2D and 3D imaging of live or fixed cancer cell lines and a wide range of primary cells, at the same time pair well with various fluorescent markers. PK Mem dyes can be applied to neuronal imaging in brain slices and in vivo two-photon imaging. The gentle nature of PK Mem palette enables ultralong-term recording of cell migration, cardiomyocyte beating, spermiogenesis, and axonal growth cone dynamics, which are prohibitively challenging using traditional PM dyes. Notably, PK Mem dyes are optically compatible with STED/SIM imaging, which can handily upgrade the routine of time-lapse neuronal imaging, such as growth cone tracking and mitochondrial transportations, into nanoscopic resolutions.

Keywords: long-term imaging; membrane dyes; neuronal imaging; phototoxicity; super-resolution imaging.