Fluorescence imaging of intracellular proteins is often achieved by using transfection-induced expression of fluorescent protein. This can potentially impose artifacts such as loss of function or over-expression of target proteins. Direct labeling of the intracellular protein is an alternative to transfection, but is largely limited by permeability of the fluorescent probes. Here, we have developed a high-throughput technique for labeling intracellular proteins of living cells. The technique makes use of Streptolysin O (SLO), a bacterial enzyme that permeabilizes cells to DNA, RNA, proteins. We show that SLO can be used to deliver a variety of fluorescent probes; ranging from organic dyes (<1 kDa in size) to fluorescent immunoglobulin antibody (~150 kDa) for specific labeling of intracellular proteins. We demonstrate in numerous ways that after permeabilization the cells remained viable and responded normally to cell signaling protein. We applied this technique to observe the dynamic motion of labeled actin and mitochondria using super-resolution fluorescence microscopy (dSTORM). Furthermore, we show the ability to image single proteins inside the living cell by tracking single molecules of kinesin using photostable probes
