单分子定位超分辨显微成像有机荧光探针的研究进展

在生物医学领域,对纳米尺寸级别的微小生物目标进行精确定位研究具有非常重要的意义,而光学显微成像技术为此提供了强有力的工具。 光学显微成像技术受到光学衍射极限的限制,难以分辨尺寸在衍射极限(<200 nm)以下的生物结构,无法直接获取微小生物结构信息,阻碍了生物医学的进一步发展。 近年来,随着纳米分辨显微成像技术的出现,新型荧光探针的开发、成像系统与设备的不断发展及成像算法不断完善地深入结合,促进了光学衍射极限以下尺寸微观目标的研究。 基于单分子定位的超分辨荧光显微成像(SMLM)包括光激活定位成像(PALM)与随机光学重构超分辨成像(STORM),将有机荧光探针与超分辨光学显微成像技术紧密结合在一起,荧光探针的光物理性质直接决定着超分辨成像结果的好坏。 因此,设计不同性能的荧光探针可以实现超精细结构的不同超分辨成像,为研究其生物学功能提供了有力的工具。 本文着重围绕基于SMLM的原理、有机荧光探针的设计要求、用于SMLM的荧光探针种类及其生物应用等方面进行总结综述,指出了单分子定位成像上存在的不足,并对其发展方向进行了展望,希望为对超分辨成像研究感兴趣或初涉该领域的研究者提供成像理论与探针设计方面的帮助。     

新型不对称五甲川菁染料的合成及光稳定性能研究

合成并表征了5种不对称五甲川菁染料,染料在甲醇中的最大吸收和荧光光谱在646—666nm之间.光降解实验证明两端取代基结构呈不对称的染料,其光稳定性明显高于两端取代基结构对称的染料.染料荧光光谱和pH值的关系表明,染料中引入苯环取代基可以增强染料在酸性或碱性溶液中的稳定性.     

苯乙烯类菁染料pH探针的合成与活细胞成像

合成了一个以苯乙烯类菁染料为母体的含胺类基团的pH荧光探针, 合成方法简单, 产物收率高, 提纯方便. 在乙醇-水体系中的性能测试结果表明, 随着pH值的降低, 其非质子化形态的吸收峰强度逐渐降低, 而质子化形式的长波长吸收峰强度逐渐升高, 长、短波长相差(Δλ)130 nm, 颜色由黄色变成粉红色, 利于可视观察; 探针在氨基呈非质子化形式时没有荧光, 氨基质子化后探针的荧光强度显著增强. 活细胞实验表明, 探针可以穿透细胞膜, 在细胞质内pH值偏低区域显示红色荧光, 可以用来定性探测酸性细胞器.     

五甲川菁染料敏化SnO_2纳光结构多孔膜电极的光电化学研究

研究了五甲川菁敏化SnO_2纳米结构电极的光电化学行为.结合循环伏安曲线图及五甲川菁的光吸收阈值,初步确定五甲川菁染料电子基态和激发态能级位置.结果表明,五甲川菁染料电子激发态能级位置能与SnO_2纳米粒子导带边位置相匹配,因而使用该染料敏化可以显著地提高SnO_2纳米结构电极的光电流,使SnO_2纳米结构电极吸收波长红移至可见光区和近红外区,光电转换效率得到明显改善,IPCE值(单色光的转换效率)最高可达45.7％.     

基于Cy7类菁染料分子探针的设计策略

菁染料是一类经典的荧光染料母核, 具有摩尔消光系数大、 吸收波长可调、 溶解性良好及生物兼容性好等优点, 被广泛用于蛋白标记、 痕量金属离子检测、 生物活性物质检测、 细胞和活体成像及肿瘤靶向治疗等领域. 近年来, 生物医学领域对活体结构及功能成像深度提出更高的需求, 基于优异的长波长染料母核开发近红外荧光分子探针逐渐成为领域的研究重点. 吲哚七甲川菁染料(Cy7)是一类最具代表性的菁染料, 本文重点综合评述了自1992年以来基于Cy7结构开发的分子探针, 并介绍了该类荧光探针的设计策略. 最后, 讨论了该领域研究面临的挑战, 并对未来的发展方向进行了总结和展望.     

五甲川菁染料敏化SnO~2纳米结构多孔膜电极的光电化学研究

研究了五甲川菁敏化SnO~2纳米结构电极的光电化学行为。结合循环伏安曲线图及五甲川菁的光吸收阈值,初步确定五甲川菁染料电子基态和激发态能级位置。结果表明,五甲川菁染料电子激发态能级位置能与SnO~2纳米粒子导带边位置相匹配,因而使用该染料敏化可以显著地提高SnO~2纳米结构电极的光电流,使SnO~2纳米结构电极吸收波长红移至可见光区和近红外区,光电转换效率得到明显改善,IPCE值(单色光的转换效率)最高可达45.7%。     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

新型水溶性不对称五甲川吲哚菁染料的合成、荧光性能及荧光标记

合成了枝状聚乙二醇醚链取代的新型不对称五甲川吲哚菁荧光染料,利用1H NMR、HRMS等技术手段表征了化合物的结构,并测定了染料的荧光性能、光稳定性,标示了牛血清白蛋白。结果表明,该染料的最大吸收波长为657 nm,最大荧光发射波长为671 nm,荧光量子产率为0.24,经过8 h的光照反应,染料有4.4%产生光降解,溶于水,n(染料)∶n(牛血清白蛋白)=2∶1时,标记蛋白质的染料/蛋白质(D/P)值达1.57。     

一种耐光漂白的碳菁型pH荧光探针的合成与细胞成像

设计合成了一种引入多个氯原子取代的耐光漂白的碳菁型pH荧光探针(CyCl4), 在500 nm波长光的激发下, 该探针具有530和596 nm两个波长的荧光峰, 其中的596 nm荧光峰在pH 8.5环境中具有最高的荧光信号值, 在pH<8.1和>9.8的条件下则荧光很弱, 通过密度泛函方法计算这三种荧光状态分别对应的三种构象结构A, B和C, 而结构B中的两个磺酸基构成分子内氢键而具有共面性几何构型, 共轭程度最好, 因而其荧光发射能力最强. 测量了该探针的荧光量子产率、瞬态荧光光谱和耐光漂白性能, 其耐光漂白性能强于一般染料型荧光探针. 最后将该探针应用于前列腺癌活细胞荧光成像, 并发现其在细胞膜表面的聚集现象.     

一种含聚乙二醇非离子亲水基团的新型水溶性荧光染料的合成及细胞成像

通过向吲哚环“N”位上引入一个含聚乙二醇(PEG)醚链的非离子亲水基团,合成了一种新型水溶性不对称五甲川吲哚菁染料。 用核磁(¹H NMR)和高分辨质谱(HRMS)表征染料的结构,测试了染料的光谱性能,标记牛血清白蛋白(BSA),并对固定细胞和活细胞分别染色。 结果表明,该染料在水中的最大紫外吸收波长和荧光发射波长分别为648和668 nm,斯托克斯(stokes)位移为20 nm,荧光量子产率(Ф)为0.13,以碘钨灯为光源光照8 h后,染料光降解率为5.8%。 用染料的NHS活性酯标记牛血清白蛋白,标示率(D/P)为1.16。 对固定细胞染色发现,染料可对细胞整体着色,对细胞核染色最明显,能清晰看到核仁。 对活细胞染色发现,有少量染料跨膜进入细胞内部,对细胞质和细胞核有微弱染色,但在活细胞膜上聚集明显。     

Quantitative Assessment of Labeling Probes for Super-Resolution Microscopy Using Designer DNA Nanostructures

Improving labeling probes for state-of-the-art super-resolution microscopy is becoming of major importance. However, there is currently a lack of tools to quantitatively evaluate probe performance regarding efficiency, precision, and achievable resolution in an unbiased yet modular fashion. Herein, we introduce designer DNA origami structures combined with DNA-PAINT to overcome this issue and evaluate labeling efficiency, precision, and quantification using antibodies and nanobodies as exemplary labeling probes. Whereas current assessment of binders is mostly qualitative, e. g. based on an expected staining pattern, we herein present a quantitative analysis platform of the antigen labeling efficiency and achievable resolution, allowing researchers to choose the best performing binder. The platform can furthermore be readily adapted for discovery and precise quantification of a large variety of additional labeling probes.     

Addressing the Requirements of High‐Sensitivity Single‐Molecule Imaging of Low‐Copy‐Number Proteins in Bacteria

Single‐molecule fluorescence super‐resolution imaging and tracking provide nanometer‐scale information about subcellular protein positions and dynamics. These single‐molecule imaging experiments can be very powerful, but they are best suited to high‐copy number proteins where many measurements can be made sequentially in each cell. We describe artifacts associated with the challenge of imaging a protein expressed in only a few copies per cell. We image live Bacillus subtilis in a fluorescence microscope, and demonstrate that under standard single‐molecule imaging conditions, unlabeled B. subtilis cells display punctate red fluorescent spots indistinguishable from the few PAmCherry fluorescent protein single molecules under investigation. All Bacillus species investigated were strongly affected by this artifact, whereas we did not find a significant number of these background sources in two other species we investigated, Enterococcus faecalis and Escherichia coli. With single‐molecule resolution, we characterize the number, spatial distribution, and intensities of these impurity spots.     

Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy**

Formylation of 2,6-dichloro-5-R-nicotinic acids at C-4 followed by condensation with 3-hydroxy-N,N-dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with “small molecules” provided specific labeling (covalent and non-covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two-color STED microscopy with a 775 nm STED laser.     

Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.     

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45–55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.     

一种新型咔唑类菁染料黏度荧光探针

合成了一种带有醛基的咔唑类菁染料KQ, 其自身的荧光量子产率较低, 对极性的敏感性小, 且荧光信号不受核酸等生物分子的干扰. KQ对环境黏度有很好的荧光响应, 相对荧光强度随着环境黏度的增大而增强, 并且在1×10^-3~1.3216 Pa·s黏度范围内, 染料的荧光强度与溶液的黏度呈良好的线性关系. 活细胞荧光成像实验结果表明, 染料KQ具有良好的细胞膜通透性, 并可对细胞内不同位置的黏度检测成像.     

Scanning Probe Microscopy beyond Imaging: A General Tool for Quantitative Analysis

A simple, fast and general approach for quantitative analysis of scanning probe microscopy (SPM) images is reported. As a proof of concept it is used to determine with a high degree of precision the value of observables such as 1) the height, 2) the flowing current and 3) the corresponding surface potential (SP) of flat nanostructures such as gold electrodes, organic semiconductor architectures and graphenic sheets. Despite histogram analysis, or frequency count (Fc), being the most common mathematical tool used to analyse SPM images, the analytical approach is still lacking. By using the mathematical relationship between Fc and the collected data, the proposed method allows quantitative information on observable values close to the noise level to be gained. For instance, the thickness of nanostructures deposited on very rough substrates can be quantified, and this makes it possible to distinguish the contribution of an adsorbed nanostructure from that of the underlying substrate. Being non‐numerical, this versatile analytical approach is a useful and general tool for quantitative analysis of the Fc that enables all signals acquired and recorded by an SPM data array to be studied with high precision.     

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.