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.
RNAs play pivotal roles in many biological processes, such as translation, gene editing etc. Since RNAs are usually smaller in size than optical diffraction limit, it is impossible for optical microscopes to get deep insight of them. With the recent emergence of super-resolution optical imaging microscopies, it becomes possible to study the ultra-fine structure of such RNA in side cells. Different kind of fluorescent probes are used for RNA labeling and imaging inside cells. However, most of them cannot be used for live-cell super-resolution imaging, due to typical laser toxicity to live cells. Herein, we developed a new type of RNA-labeled probe, which can be used for STED imaging of RNA in living cells. We believe this probe is of great significance for studying biological behaviors of RNA in living cells.
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.
Unfolded or misfolded protein accumulation inside Endoplasmic Reticulum (ER) will cause ER stress and subsequently will activate cellular autophagy to release ER stress, which would ultimately result in microviscosity changes. However, even though, it is highly significant to gain a quantitative assessment of microviscosity changes during ER autophagy to study ER stress and autophagy behaviors related diseases, it has rarely been reported yet. In this work, we have reported a BODIPY based fluorescent molecular rotor that can covalently bind with vicinal dithiols containing nascent proteins in ER and hence can result in ER stress through the inhibition of the folding of nascent proteins. The change in local viscosity, caused by the release of the stress in cells through autophagy, was quantified by the probe using fluorescence lifetime imaging. This work basically demonstrates the possibility of introducing synthetic chemical probe as a promising tool to diagnose ER-viscosity-related diseases.