为实现无标记样本的定量检测，借助NX12.0搭配相关器件自主设计了一款小型化相位显微镜。相较于市面上售价高昂的相位显微镜，所设计显微镜无需相干器件，且在满足系统分辨率的前提下，将体积缩小了约60%，大幅提高了便携性，同时成本仅需5000元左右。系统中还嵌入了自动对焦算法和基于变换域的视场校正算法，以加快检测速度和相位恢复的准确率。经测试，系统在10倍物镜下的分辨率达到了分辨率板极限2.19 μm，同时随机相位板的检测表明相位恢复的准确率也达到了基本需求。除此之外，还对活细胞结构和平面玻璃的缺陷进行了测试。结果表明，所提系统不仅能对活细胞进行较好的定量测量，还可以在透明/半透明平面缺陷的检测中发挥重要作用，更是证明了这种成本低、便携性高、可实现无标记样本定量的系统设计方案的可行性。

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 30 s and demonstrate the same precision as manual image analyses.

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 e±ciency (ET 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.

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 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield (PLQY) was 64%. The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8mW. The lateral resolution of 42.0 nm 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 3.1 ns, consistent with the in vitro value, suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.

针对现有表面等离子激元折射率传感器纵向探测深度小、探测范围无法覆盖整个细胞厚度的问题,提出一种大探测深度、高灵敏度的活细胞折射率实时测量方法,并利用该方法开展了药物敏感性的实验研究。基于偏振选择吸收效应,设计并搭建了全内反射条件下的石墨烯折射率传感系统,进行了不同质量分数氯化钠溶液折射率的测量,结果表明系统具有9.5×10 ⁶ mV/RIU的灵敏度和5.5×10 ^-7RIU的分辨率;利用该系统开展了活细胞药物敏感性的实验研究,分别研究了顺铂和紫杉醇作用于Ramos细胞和Jeko-1细胞时生物演化过程中细胞折射率的实时变化规律,验证了折射率变化与其药性机理作用的一致性。

本文发展了一种针对于固定FRET质粒的单波长激发的E-FRET方法(SDW-E-FRET)。相比于E-FRET方法, SDW-E-FRET只需要测量供体激发时供体通道的荧光强度(IDD)和FRET通道的荧光强度(IDA), 因此该方法可以实现活细胞的快速定量FRET成像。结合双通道荧光显微成像系统, 该方法无需任何机械切换, 定量FRET成像的速度只取决于CCD相机的成像速度, 因而特别适合活细胞实时动态定量FRET成像。在本研究小组发展的双通道荧光显微镜平台上, 应用SDW-E-FRET方法测量了C5V和C17V的FRET效率, 得到了与其它方法测量的一致的结果。

细胞内的pH是细胞内多种酶活性和生理活动的重要调节因素, 准确、动态的监测细胞内pH变化对研究细胞内的活动至关重要。一些荧光小分子可以感应pH的变化, 同时具有较高的灵敏度和特异性, 对细胞损伤较小且标记操作简单, 已逐渐发展成为一种监测细胞内pH变化的有效方法。本文主要介绍目前常用pH敏感的荧光探针及其在活细胞研究中的进展。

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.