Fabrication And Application Of FISH Probes For Optical Super Resolution Imaging Of Telomeres

Telomere is a kind of special DNA sequence,located at the terminal of eukaryotic chromosomes.During the DNA replication,the end of the chromosome can not be replicated,so telomeres are gradually shortened with periodic replication,which limits the aging of normal cells and is closely related to diseases.Hence,telomere-research is of great significance in cancer treatment and life sciences.Fluorescence in situ hybridization(FISH)is one of the widely used technologies in telomere-research.Fluorescence microscope is always used to observe samples,whose imaging resolution is affected by the optical diffraction limit,usually about 200~300 nm. This resolution makes it impossible for people to study the telomeres carefully.In this paper,we have proposed to combine FISH with single molecule location microscope(SMLM),an optical super resolution imaging microscope,for high-resolution imaging of telomeres.A simple,inexpensive and efficient protocol of making FISH probes has been designed and realized by using Cd SSe/Zn S quantum dots(QDs)fabricated with telomere complementary DNA strands.SMLM is also used to study the image of telomeres labeled with FISH probes.The resolution of imaging breaks the diffraction limit and obtains the precise location of telomere,and lays a foundation for telomere-associated cancer treatments.Main results include:At first,the Cd SSe/Zn S QDs(maximum ?em=640nm)in the oil phase must be transferred into the aqueous phase,then FISH probes have been prepared by decorating QDs with DNA strands specific to telomeres.Characterization of the optical properties of QDs and FISH probes,we can conclude that,this method is not only has no great influence on the excellent fluorescence property of QDs,but also avoids the hybridization between the primers caused by the annealing treatment of the general organic dye probes,and then affects the accuracy of the imaging.Next,telomeres labeled with FISH probes have been observed by confocal microscopy.We have verified the feasibility of the probes for FISH experiments via the confocal images of samples. In addition,the PNA probes were first hybridized with telomeres,and then FISH probes,which further confirmed the specificity of the FISH probes.But we also found the disadvantage of the conventional fluorescence microscope in observing telomeres,that is,it is impossible to achieve higher resolution,which limits the further study of telomeres.As a result,a protocol based on FISH and SMLM for high-resolution imaging of telomeres has been designed.Last,super-resolution image(SRI)was used to investigate the telomeres with the FISH probes.Though the SMLM image and the localization precision of telomeres,we can conclude that telomeres are very clear in SMLM images and the resolution is about 30 nm,which solves the problem and acquires accurate location of the telomeres.The hybridization time of the FISH probes was also studied here,and the optimal parameter was around 6~12 h.Besides,compared with SMLM images of organic dyes(e.g.Tel C-Alexa Fluor 488),we found several merits of FISH probes: First,fluorescence blinking of QDs probe needs only one excitation light(i.e.405nm).While fluorescence blinking of the organic fluorophores usually requires two illumination lights,that is,the activation light(i.e.405nm)and the imaging light.Second,the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol,which is a key buffer component for organic fluorophores' blinking.Third,multiple switching times and a good optical stability make the QDs more suitable for long-term imaging.Last but not least,the lower-cost QDs probes can be used to specifically image other targets(e.g.cells,organelles,proteins,etc.)by replacing DNA strands with other ligands,hence the QDs FISH probes possess a good universality.To sum up,QDs FISH probes and SMLM have enabled new insights into telomeres on the molecular level,and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.They will have a good prospect in cell imaging,biomedicine,and diagnosis of tumors.