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《中国化学快报》2022,33(12):5042-5046
The need for temporal resolution and long-term stability in super-resolution fluorescence imaging has motivated research to improve the photostability of fluorescent probes. Due to the inevitable photobleaching of fluorophores, it is difficult to obtain long-term super-resolution imaging regardless of the self-healing strategy of introducing peroxide scavengers or the strategy of fluorophore structure modification to suppress TICT formation. The buffered fluorogenic probe uses the intact probes in the buffer pool to continuously replace the photobleached ones in the target, which greatly improves the photostability and enables stable dynamic super-resolution imaging for a long time. But the buffering capacity comes at the expense of reducing the number of fluorescent probes in targets, resulting in low staining fluorescence intensity. In this paper, we selected BODIPY 493, a lipid droplet probe with high fluorescence brightness, to explore the dynamic process of lipid droplet staining of this probe in cells. We found that BODIPY 493 only needs very low laser power for lipid droplet imaging due to the high molecular accumulation in lipid droplets and the high brightness, and the spatiotemporal resolution is greatly improved. More importantly, we found that BODIPY 493 also has a certain buffering capacity, which enables BODIPY 493 to be used for super-resolution imaging of lipid droplet dynamics. This work reminds researchers to coordinate the buffering capacity and brightness of fluorogenic probes. 相似文献
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Delia Bernik Elizabeth Tymczyszyn Marta Edit Daraio R. Martín Negri 《Photochemistry and photobiology》1999,70(1):40-48
Abstract— Fluorescence emission from merocyanine 540 (MC540) dimers was observed in dipalmitoylphosphatidylcholine (DPPC) vesicles. This unusual behavior was observed only for vesicles in the gel-phase state. No dimer fluorescence was observed either in monopalmitoylphosphati-dylcholine (C16 PC) micelles or in liquid-crystalline DPPC vesicles, indicating that dimer fluorescence efficiency increases in highly packed interfaces. The excitonic theory of Kasha was used to interpret the spectral features. The overall fluorescence quantum yield (φr ) decreases with decreasing lipid: probe ratio, not only because of the presence of a weakly fluorescent dimer that absorbs a high fraction of the total absorbed light but also due to quenching of monomer emission. This suggests the existence of probe domains. The dimer fluorescence quantum yields (φm ) were estimated in DPPC large unilamellar vesicles (LUV) and DPPC multilamellar vesicles. The dependence of φr with probe concentration is compatible with values of φm lower than 0.05. The dimerization equilibrium of MC540 in C16 PC micelles and DPPC-LUV was also studied. Apparent dimerization equilibrium constants, Kdapp and dimer absorption spectrum were calculated in C16 PC micelles for the first time. The dimerization equilibrium constant in DPPC-LUV was calculated and discussed in terms of the fraction of volume occupied by the lipid phase. 相似文献
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现有的光学超分辨显微成像技术主要依赖于特殊的荧光标记物,其对于大多数非荧光样品的超分辨成像就变得无能为力。因此我们提出将光学相减显微技术应用到非荧光样品的成像当中,利用普通共聚焦光斑和面包圈型光斑分别激发样品的散射光成像,从而得到样品同一区域的两幅图像,再通过图像相减的方法提高了图像空间分辨率。不同于一般的超分辨成像方法,这种光学相减显微镜不需要特殊的样品预处理过程,同时两次成像的激发光强度可以保持在一个较低水平,避免了样品损伤的影响。随后金纳米小球和有机聚合物微丝的散射成像实验证明了光学相减显微镜可以将空间分辨率提高到215 nm (0.33λ, 1λ = 650 nm),并且通过探测散射信号得到更多的样品细节信息。 相似文献
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Dr. Xinfu Zhang Dr. Lingcheng Chen Zhenlong Huang Ni Ling Prof. Yi Xiao 《Chemistry (Weinheim an der Bergstrasse, Germany)》2021,27(11):3688-3693
Newly emerging super-resolution imaging techniques provide opportunities for precise observations on cellular microstructures. However, they also impose severe demands on fluorophores. Here, we develop a new series of NIR xanthene dyes, named as KRh s, by replacing the 10-position O of rhodamines with a cyclo-ketal. KRh s display an intense NIR emission peak at 700 nm with fluorescence quantum yields up to 0.64. More importantly, they, without the aid of enhancing buffer, exhibit stochastic fluorescence off–on switches to support time-resolved localization of single fluorophore. KRh s are functionalized into KRh-MitoFix , KRh-Mem and KRh-Halo that demonstrate mitochondria, plasma membrane and fusion protein targeting ability, respectively. Consequently, these KRh probes demonstrate straightforward usage for super-resolution imaging of these targets in live cells. Therefore, KRh s merit future development for fluorescence labeling and super-resolution imaging in the NIR region. 相似文献
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Min Zhang Jing Gao Junling Chen Mingjun Cai Junguang Jiang Zhiyuan Tian Hongda Wang 《中国科学:化学(英文版)》2016,59(7):848-852
Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes or proteins as fluorophores. The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy’s localization precision and imaging resolution. Fluorophores TAMRA and Atto Rho6G, which can interact with macrocyclic host cucurbit[7]uril (CB7) to form host-guest compounds, were found to improve the fluorescence intensity and lifetimes of these dyes. We enhanced the localization precision of direct stochastic optical reconstruction microscopy (dSTORM) by introducing CB7 into the imaging buffer, and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6G increase over 2 times. 相似文献
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简要梳理了超分辨荧光共聚焦显微成像技术的发展历史.在此基础上, 重点总结了中国自主创新高分辨荧光共聚焦显微成像技术取得的突破, 特别是2006年以来, 中国科学院和北京大学成功研发了几款较为成熟的超分辨荧光共聚焦显微系统, 有力地推动了中国自主创新仪器研制的进展.最后, 在大型仪器设备开放共享评价考核数据分析的基础上, 着重分析了高校和科研院所激光共聚焦显微镜的开放共享使用情况. 相似文献
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Andrew T. Frawley Virginia Wycisk Yaoyao Xiong Silvia Galiani Erdinc Sezgin Iztok Urban
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Andreas Vargas Jentzsch Kathryn G. Leslie Christian Eggeling Harry L. Anderson 《Chemical science》2020,11(33):8955
Dyads consisting of a photochromic switch covalently linked to a fluorescent dye allow the emission from the dye to be controlled by reversible photoisomerization of the switch; one form of the switch quenches fluorescence by accepting energy from the dye. Here we investigate the use of dyads of this type for super-resolution imaging of lipid bilayers. Giant unilamellar vesicles stained with the dyads were imaged with about a two-fold resolution-enhancement compared with conventional confocal microscopy. This was achieved by exciting the fluorophore at 594 nm, using a switch activated by violet and red light (405/640 nm).A photoswitchable quencher can be used to reversibly turn off the emission from a fluorescent dye, generating a small molecule dyad that is effective for super-resolution RESOLFT microscopy. 相似文献
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Xiaolian Li Jiazhu Zheng Wenjuan Liu Qinglong Qiao Jie Chen Wei Zhou Zhaochao Xu 《中国化学快报》2021,31(11):2937-2940
Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells. 相似文献
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《中国化学快报》2020,31(11):2937-2940
Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells. 相似文献
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It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8+/-0.1 and 7.4+/-0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0-2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8+/-0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00-0.30 mol l(-1)). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01-0.30 mol l(-1). The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study. 相似文献
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Dr. Seong Ho Kim Prof. Dr. Isaac T. S. Li 《Angewandte Chemie (International ed. in English)》2023,62(7):e202217028
DNA-PAINT enabled super-resolution imaging through the transient binding of fluorescently-labelled single-stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB-PAINT provides localization precision similar to traditional linear imager DNA-PAINT. We also show that MB-PAINT is ideally suited for fast super-resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free-diffusing imagers in traditional DNA-PAINT at the cell-substrate interface. 相似文献
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HOU ShangGuo LIANG Le DENG SuHui CHEN JianFang HUANG Qing CHENG Ya FAN ChunHai 《中国科学:化学(英文版)》2014,57(1):100-106
Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of<200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods. 相似文献
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Pedro F. Aramendia Marianne Krieg Christian Nitsch Edith Bittersmann Silvia E. Braslavsky 《Photochemistry and photobiology》1988,48(2):187-194
Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> øT > 0.02 in ethanol and 0.01 > øT - > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay. 相似文献
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Grunwald C Schulze K Giannone G Cognet L Lounis B Choquet D Tampé R 《Journal of the American Chemical Society》2011,133(21):8090-8093
Single-molecule applications, saturated pattern excitation microscopy, and stimulated emission depletion (STED) microscopy demand bright as well as highly stable fluorescent dyes. Here we describe the synthesis of quantum-yield-optimized fluorophores for reversible, site-specific labeling of proteins or macromolecular complexes. We used polyproline-II (PPII) helices as sufficiently rigid spacers with various lengths to improve the fluorescence signals of a set of different trisNTA-fluorophores. The improved quantum yields were demonstrated by steady-state and fluorescence lifetime analyses. As a proof of principle, we characterized the trisNTA-PPII-fluorophores with respect to in vivo protein labeling and super-resolution imaging at synapses of living neurons. The distribution of His-tagged AMPA receptors (GluA1) in spatially restricted synaptic clefts was imaged by confocal and STED microscopy. The comparison of fluorescence intensity profiles revealed the superior resolution of STED microscopy. These results highlight the advantages of biocompatible and, in particular, small and photostable trisNTA-PPII-fluorophores in super-resolution microscopy. 相似文献
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Ariola FS Mudaliar DJ Walvick RP Heikal AA 《Physical chemistry chemical physics : PCCP》2006,8(39):4517-4529
Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions. 相似文献
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Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells. 相似文献
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Prof. Weijie Chi Dr. Davin Tan Prof. Qinglong Qiao Prof. Zhaochao Xu Prof. Xiaogang Liu 《Angewandte Chemie (International ed. in English)》2023,62(39):e202306061
Single-molecule localization microscopy (SMLM) has found extensive applications in various fields of biology and chemistry. As a vital component of SMLM, fluorophores play an essential role in obtaining super-resolution fluorescence images. Recent research on spontaneously blinking fluorophores has greatly simplified the experimental setups and extended the imaging duration of SMLM. To support this crucial development, this review provides a comprehensive overview of the development of spontaneously blinking rhodamines from 2014 to 2023, as well as the key mechanistic aspects of intramolecular spirocyclization reactions. We hope that by offering insightful design guidelines, this review will contribute to accelerating the advancement of super-resolution imaging technologies. 相似文献
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Fluorescence imaging is an important branch of bioimaging. It is non-invasive and provides superior spatial and temporal resolution during the real-time monitoring of biological samples of interest. Although the spatial resolution limit of optical microscopes is about 200 nm, due to the diffraction limit, with the application of super-resolution fluorescence microscopy technologies this limit has been pushed below 30 nm. This makes it feasible to visualize biological structures in subcellular levels and to monitor subcellular biological processes in real time. However, due to the complexity of the biological structure and components within cells, simultaneous staining and monitoring multiple intracellular components with different coloured fluorophores is often needed during multiplex imaging, to better understand biological processes. Aggregation-induced emission luminogens(AIEgen) and AIEgen based nanoparticles(NPs) have presented many advantages in fluorescence imaging, with strong potential for biological science and nano-medicine. Herein this review, we focus on the advantages of AIEgen and AIEgen NP in cell-based fluorescence imaging, and the latest advances of AIEgens in cell-based multiplex imaging are summarized and discussed. The future perspectives are proposed. 相似文献