Study on a novel probe for stimulated emission depletion Super-resolution Imaging of Mitochondria

Optical microscopy has the advantages of real-time, non-invasive, tomography, three-dimensional imaging and living imaging. However, its spatial resolution cannot exceed half wavelength due to the existence of optical diffraction limit, which limits the development of optical microscopy. The primary task of super-resolution imaging is to break the diffraction limit and improve the resolution of optical microscopy for study of subcellular structure. Many kinds of super-resolution imaging technologies have been reported, among which the stimulated emission depletion (STED) microscopy is the earliest imaging technology to break the optical diffraction limit at present. STED microscopy can achieve nanometer-scale spatial resolution by breaking the optical diffraction limit with pure optical methods and a clever optical design. However, the application of STED microscopy in biomedicine, especially in live cell imaging is limited by high illumination power of STED light. In this paper, a new type of STED probe has been developed. The spectral analysis results show that the peak of the excitation and emission spectrum of this probe is as far as 122 nm away from each other, which is very suitable for the study of STED super-resolution because of its long stokes redshift. After colocalization with commercial mitochondrial dyes, it was found that the probe had a higher localization coefficient with commercial dyes and could be well positioned on mitochondrial organelles. At the same time, it was found that strong mitochondrial signal could be detected with low-power excitation light (only 1 mu W in the experiment), and can get higher resolution of 62 nm under the STED light with 39.5 mW. The result of measuring the transverse resolution obtained by STED light under different power shows that the saturated light power of the probe is 3.5 mW (1.1 MW.cm(-2)). Through the anti-bleaching testing, the probe still has a strong fluorescence intensity after more than 300 times of high power light irradiation, which indicates that the probe has a strong anti-bleaching property. Through a series of tests, this paper present a novel STED probe which has good mitochondrial targeting, excellent photobleaching-resistance, high resolution and low saturation power, which provides a new research tool for long-term live cell mitochondrial super-resolution imaging.