Intracellular Delivery of Membrane Impermeable Photostable Fluorescent Probes into Living Cells for Super-Resolution Microscopy

Specific labeling of the cellular target with fluorophore is one of the fundamental requirement in all fluorescence imaging techniques including single molecule and super-resolution microscopy techniques. While extracellular targets may be tagged with virtually any kind of probes, intracellular labeling of living cells is limited to the use of fluorescent proteins and limited selection of membrane permeable dyes. Here we show that pore forming proteins can be used to temporarily permeabilize the cells and allow delivery of various fluorescent probes, ranging from organic dyes (<1 kDa) to fluorescent immunoglobulin antibody (∼150 kDa), for specific labeling of intracellular targets for live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear translocation of nanobody-labeled p65 proteins in response to chemical stimulation. One of the most photostable but membrane impermeable organic fluorophore, Atto 647N, has been delivered into cells to label actin fibers and kinesin dimers and to perform super-resolution fluorescence microscopy imaging dSTORM and single molecule tracking, respectively. This technique opens up a large number of stable, but otherwise membrane impermeable fluorescence labeling probes that are available for investigating transfected and endogenous intracellular targets.