Organic Fluorescent Probes For Three Types Of Subcellular Structures In Live-cell Super-resolution Imaging

The traditional optical microscopy cannot obtain spatial resolution below 200 nm because of the optical diffraction limit.In the past decade,scientists developed new techniques called super-resolution fluorescence microscopy to work around the supposed limit,and the Nobel Prize in chemistry in the year of 2014 was awarded to those who made outstanding contributions to these techniques based on different strategies.There are three main factors influencing the imaging quality and spatiotemporal resolution of super-resolution fluorescence microscopy,which can be summed up in optical hardware system,super-resolution algorithm and the labeling of biological samples.Fast development of labeling methods,especially organic fluorescent probes which efficiently improved the resolution of super-resolution imaging,has been achieved in the past ten years.Many existing commercially available fluorescent dyes are famous for their excellent optical properties,but they cannot be transported directly into living cells because of its low permeability,thus hindering their applications in live-cell super-resolution imaging.In order to solve this problem,we designed and synthesized two types of organic fluorescent probes for microfilament and lysosomes in live cells,respectively.We also developed a commercially available dye as a new live-cell probe for mitochondria with very excellent optical properties.Based on the excellent properties of the above probes,we applied them in super-resolution imaging,and discovered new phenomenons about lysosomes and mitochondria in live cells.The main contents are shown as follows:(1)We designed and synthesized organic fluorescent probes for microfilament and lysosomes in live cells.The structures of these probes include two parts.The first part consists of recognition units and cell-penetrating peptides,while the other part contains commercially available fluorescent dyes,regardless of their membrane permeability.Utilizing the excellent super-resolution properties of commercially available dyes,this new design has the advantage of cost saving and flexibility in choosing dyes.We worked out the optimal experimental condition of the probes and applied these probes in different types of live cells with distinct colors.(2)We determined the performance of these probes mentioned above in stochastic optical reconstruction microscopy(STORM)and structured illumination microscopy(SIM).Using the probe Actin-647 in STORM,we resolved the microfilament in live cells at 60-nm spatial resolution and 10-s temporal resolution.Meanwhile,we observed the uneven distribution of cysteine cathepsins in lysosomes and a dynamic process of lysosomal fission and fusion in live-cell SIM imaging using the probe Lysosome-565.(3)Based on the commercially available dye Atto 647 N,we developed it as a new live-cell probe for mitochondria,which has the best brightness and anti-bleaching ability among the existing mitochondrial probes.Using Mito-647 N and Lysosome-565 in lorg-term SIM,we recorded the 13-min lysosome-mitochondrion interactions at spatial resolution of approximately 90 nm for the first time in live cells.The successful application of these probes in super-resolution imaging,especially the improvement of imaging resolution and the discovery of the new phenomena of mitochondria and lysosomes,proved the significance of the introduction of commercially available dyes into the probes for live-cell super-resolution imaging.