Stimulated emission depletion (STED) microscopy enables visualization of previously indiscernible subcellular structures in biological cells. However, it is costly and complicated to built a STED microscope system because of the dependence on the depletion laser, especially in multicolor imaging. In addition, inefficient fluorescence inhibition on account of the small stimulated emission cross-section results in a huge demand for the power of the depletion laser, which hinders its application to study living cells. Here we present a method based on phasor plot analysis for achieving two-color STED imaging at a relatively low depletion power, which is implemented in a pulsed-STED microscope system with only a pair of excitation and depletion laser beams. Firstly, two fluorescent dyes with similar spectral characteristics but different lifetimes were selected for two-color imaging. Secondly, a depletion laser with a wavelength closer to the emission maximum was applied to boost the depletion efficiency and reduce the required depletion power. This approach makes two-color STED imaging easier and has the potential to realize multi-color STED super-resolution imaging without the need of additional lasers, thereby offering more convenient and efficient service to researchers.
We propose a spatiotemporal modulation method to achieve super-resolution imaging at a depletion power two orders of magnitude lower than traditional counterpart. By increasing the pulse interval between excitation and depletion lasers, the fluorescence lifetime data contain the spatiotemporal information of confocal and STED photons at the same time. Two kinds of information are bounded by depletion pulse in a period of the pulse trains, and their intensity difference represents the stimulated emission intensity by donut-shaped depletion laser. Finally, low-power STED imaging with high image quality is realized by subtracting the enhanced stimulated emission intensity from the confocal one.
Nanoscopic optical imaging has made prominent progress in recent years, which provides a powerful tool for modern biology science. Superresolution optical imaging allows for the observation of ultra-fine structures of cells, cellular dynamics and cellular functions at nanometer scale or even single molecular level, which greatly promotes the development of life science and many other fields. However, challenges still exist for super-resolution optical imaging for live cells and thick samples in terms of imaging depth, imaging speed as well as biomedical applications. This talk will review the recent progress in superresolution optical microscopy and present our recent work. By combining stimulation emission depletion (STED) microscopy and fluorescence lifetime imaging (FLIM), a STED-FLIM superresolution microscopy was developed to improve the spatial resolution of STED and also perform FLIM imaging at nanometer resolution. A new fluorescent probe with low STED laser power was designed for live cell mitochondria imaging. STED-FLIM imaging of microtubules labeled with ATTO647N inside HeLa cells and the mitosis process was obtained, which provides new insight into the cell structure and functions. In addition, coherent adaptive optical technique (COAT) has been implemented in a stimulated emission depletion microscope to circumvent the scattering and aberration effect for thick sample imaging. Finally, stochastic optical reconstruction microscopy (STORM) superresolution imaging of mitochondrial membrane in live HeLa cells was obtained by the implementation of new fluorescent probes, improved imaging system and optimized single molecule localization algorithm. This provided an important tool and strategy for studying dynamic events and complex functions in living cells.
A series of new fluorescent probes were developed to carry out live cell super-resolution imaging with low STED laser power or suitable STORM working conditions. And STED-FLIM imaging of microtubules labeled with ATTO647N inside HeLa cells and the mitosis process was obtained, which provides new insight into the cell structure and functions.Finally, stochastic optical reconstruction microscopy (STORM) super-resolution imaging of mitochondrial membrane in live HeLa cells was obtained by the implementation of new fluorescent probes, improved imaging system and optimized single molecule localization algorithm. This provided an important tool and strategy for studying dynamic events and complex functions in living cells.