Abstract
Numerous commercial organic fluorophores with excellent optical properties are precluded from live-cell superresolution imaging due to poor cell permeability. Here, we develop a simple but effective strategy that renders cells permeable to cell-impermeable, organic fluorescent probes by using a novel peptide vehicle, PV-1. By simple coincubation with PV-1, 22 different cell-impermeable, organic fluorescent probes were efficiently delivered into live cells and specifically labeled a variety of organelles. Moreover, PV-1 can simultaneously transfer up to three different probes into live cells. By using PV-1 and these cell-impermeable fluorescent probes, we obtained multicolor, long-term, live-cell superresolution images of various organelles, which allowed us to study the dynamic interactions between them. PV-1, together with these organic fluorescent probes, will greatly broaden the applications of superresolution imaging technology in diverse live-cell studies and opens up a new avenue in the design and application of peptide vehicles.
Highlights
Precise imaging of intracellular, subcellular structures and their dynamic processes is crucial for fundamental research in biology and medicine[1,2,3,4]
Numerous commercial organic fluorophores with high fluorescence intensity and excellent photostability are precluded from live-cell structured illumination microscopy (SIM) imaging due to poor cell permeability
TP10, GALA, Penetratin (Pene), and dfTAT were selected from previous reports because they all achieved efficient cytosolic delivery of proteins or nucleic acids by simple coincubation
Summary
Subcellular structures and their dynamic processes is crucial for fundamental research in biology and medicine[1,2,3,4]. Thanks to recently developed far-field superresolution fluorescence microscopy (e.g., SIM, STED, and PALM/STORM), imaging subcellular structures with a spatial resolution beyond the diffraction limit has been achieved[4,5,6,7] Among these superresolution microscopy methods, structured illumination microscopy (SIM) is distinct in its high imaging speed and low illumination intensities; it is a standout tool for directly observing the dynamics of for long-term multicolor SIM imaging, probes. To improve the cellular uptake of organic fluorophores, several strategies have been developed, including chemical modification of fluorophores and the use of physical methods to temporarily disrupt the cell membrane, such as electroporation and nanoinjection[14,15,16] These methods present several limitations in terms of applicability and efficiency and are time-consuming and technically demanding. On the other hand, characterizing dynamic interactions between subcellular structures in live cells by SIM is difficult to achieve due to the lack of suitable livecell compatible fluorescent probes
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