Live-Cell Intracellular Storm in the Presence of Oxygen with Membrane-Impermeable Organic Fluorophores

Abstract

Live-cell super-resolution imaging is a valuable tool for studying living systems. Labeling intracellular targets inside living cells easily, quickly, and inexpensively, particularly in the presence of atmospheric oxygen, has been a challenge. Here we deliver multicolor organic dyes, which have particularly good photostability, inside cells to conduct live-cell dSTORM in the presence of oxygen. The dyes pass through temporary pores in the membrane made by a bacterial toxin Streptolysin O, with no ill effects. Intracellular targets are labelled faster (< 30 min.) with smaller amount of dyes (< 200 nM) compared to other methods, with no special equipment. Virtually any combination of protein, tagging system and dye (e.g. Nanobodies, Halo-, SNAP- and eDHFR-tag) can be applied, although the choice of dyes is important. For instance, Alexa Fluor 488 (blue), CF 568 (yellow) and Atto 655 (red) are found particularly useful. Hence the technique can also be used to label endogenous proteins, such as endogenous actin labelled by Alexa Fluor 488-Phalloidin. With proper selection of organic dyes, 2-(and 3-)color live-cell intracellular dSTORM imaging is achieved without any oxygen scavenger system.