Narrow Results

The study of the electrical coupling properties of living cells and materials plays an important role in tissue engineering and clinical applications. The electrical coupling between living cells and substrates directly affects the growth, secretion and death of cells, as well as the measurement accuracy of electrophysiological parameters. In this work, the point contact model of the electrical coupling of cells and substrates is studied. It was found that the sealing impedance and effective contact area were the most important parameters affecting the electrical coupling. Moreover, the structure of nano-scale groove was fabricated by direct laser interference patterning (DLIP), and cylindrical arrays were fabricated on resist films after exposed to electron beam lithography (EBL). The two nanostructures were used as the substrates in vitro culture. The electrical coupling properties between living cells and structural substrates were discussed subsequently. In addition, the impedance spectrum of living cells at various frequencies, the relationship between the living cell number and impedance, and the impedance of living cells at different growth periods were measured. Furthermore, the multidimensional impedance information of living cells was obtained, and the impedance deviations between the two structure arrays were analyzed to evaluate the electrical coupling properties between the living cells and substrates. The proposed multidimensional impedance analysis method provides a way for studying the electrical coupling properties of nano-structured substrates and living cells.

Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

Inorganic quantum dots (QDs) have excellent optical properties, such as high fluorescence intensity, excellent photostability and tunable emission wavelength, etc., facilitating them to be used as labels and probes for bioimaging. In this study, CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope (FLIM) and stimulated emission depletion (STED) nanoscopy imaging. The emission peak of CdSe@ZnS QDs centered at 526nm with a narrow width of 19nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400min by STED laser with 39.8mW. The lateral resolution of 42.0nm is demonstrated for single QDs under STED laser (27.5mW) irradiation. Furthermore, the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes. Meanwhile, Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM, and their fluorescence lifetime was around 3.1ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.

Exact interaction mechanism between Bax and Bcl-XL, two key Bcl-2 family proteins, is an interesting and controversial issue. Partial acceptor photobleaching-based quantitative fluorescence resonance energy transfer (FRET) measurement, PbFRET, is a widely used FRET quantification method in living cells. In this report, we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency ($E)$ images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL. The $E$ value between CFP-Bax and YFP-Bcl-XL was 4.59% in cytosol and 11.31% on mitochondria, conclusively indicating the direct interaction of the two proteins, and the interaction of the two proteins was strong on mitochondria and modest in cytosol.

Intensity-based quantitative fluorescence resonance energy transfer (FRET) is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit. This widely used technique needs complicated experimental process and manual image analyses to obtain precise results, which take a long time and restrict the application of quantitative FRET especially in living cells. In this paper, a simplified and automatic quantitative FRET (saqFRET) method with high efficiency is presented. In saqFRET, photoactivatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein (EGFP) are used to simplify FRET crosstalk elimination. Traditional manual image analyses are time consuming when the dataset is large. The proposed automatic image analyses based on deep learning can analyze 100 samples within 30s and demonstrate the same precision as manual image analyses.