A Novel Probe Delivery Strategy And Its Application In Super-resolution Imaging

The appearance of super-resolution fluorescence microscopy breaks the optical diffraction limit,which makes it possible to observe the nanoscale structure below 200 nm by fluorescence microscopy.It is very important for us to understand and reveal the physiological function of subcellular structure by using super-resolution microscopic imaging technology for fast and long-term super-resolution imaging of subcellular structure in living cells.However,the application of advanced microscope for imaging dynamic interactions between subcellular structures remain challenging,as they need fluorescent probes that not only can specifically label various subcellular structures but are also possess excellent photo stability for long-term observation.Compared with fluorescent proteins,organic fluorescent dyes have great advantages in terms of brightness and photostability,and easy to modify.However,most organic fluorescent dyes with excellent photostability and brightness are precluded from live-cell imaging due to poor cell permeability,which greatly limits the application of organic fluorescent probes in living cells.In order to improve the cellular uptake of organic fluorophores,several strategies have been developed but they have not yet achieved good results.To solve the above problems,we proposes a strategy of non-covalent delivery of fluorescent probes into living cells based on peptide vehicle.Through a series of screening,we successfully obtained a peptide carrier PV-1 which can efficiently deliver organic fluorescent probes into living cells through simple co-incubation,and verified that PV-1 has good universality in delivering fluorescent probes into living cells through 23 kinds of organic fluorescent probes with different structures.Using PV-1 and these cell-impermeable organic fluorescent probes,multi-color consecutive super-resolution images of various organelles interacting in live cells were obtained.The main contents are as follows:We proposed a labeling strategy to deliver cell-impermeable organic fluorescent probes into living cells by non-covalent binding with peptide vehicle.Through a series of screening,we successfully obtained a peptide carrier PV-1 which can efficiently deliver organic fluorescent probes into living cells through simple co-incubation.Finally,we carefully explored the specific mechanism of PV-1 delivering probes into living cells,and found that PV-1 delivered probes to living cells mainly through the macropinocytosis pathway in endocytosis.23 kinds of cell-impermeable organic fluorescent probes with different chemical structures were used to verify that PV-1 has excellent universality in delivering fluorescent probes into living cells.Further co-localization experiments proved that the use of PV-1 to deliver probes into living cells did not significantly affect the specificity of the probes.In addition,PV-1 can simultaneously deliver up to three different cell-impermeable probes into live cells,which provides a new method to label intracellular targets for the application of multicolor living cell imaging.It has been verified on a variety of super-resolution microscopies that the organic fluorescent probes with excellent optical properties delivered by PV-1 can be used for superresolution imaging of living cells.This method greatly expands the selection range of superresolution imaging probes for living cells.Using these cell-impermeable probes combined with 2D or 3D super-resolution imaging systems,we have captured the dynamic interactions between various subcellular structures.In summary,this article developed a delivery strategy that can simply and efficiently deliver cell-impermeable organic fluorescent probes into living cells.PV-1 and the fluorescent probes with excellent optical properties have achieved excellent imaging results on a variety of 2D and 3D super-resolution imaging systems,which greatly expands the range of available super-resolution imaging probes for living cells,and opens up a new avenue in the design and application of peptide vehicles.