受激辐射损耗超分辨荧光成像探针研究进展

受激辐射损耗超分辨成像可突破光学衍射极限的限制，获得纳米尺寸结构的超精细图像，荧光探针发挥了重要作用。本文主要介绍了受激辐射损耗超分辨显微成像的相关概念，包括基础光学概念、受激辐射损耗超分辨成像原理、成像系统，总结了受激辐射损耗超分辨成像荧光探针及其应用等研究进展，相信本工作能帮助化学、化工领域相关工作者了解受激辐射超分辨成像研究及其生物成像应用，尤其是可以用于该成像技术的荧光探针的相关知识，为设计、制备有效的受激辐射超分辨荧光探针提供设计思路。本文为荧光探针在生物医学光学领域的应用提出了新的需求与机遇，为化学、化工领域相关研究方向与光学成像领域的深度交叉与融合提供了新的发展契机。     

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.     

Dyes derived from benzo[a]phenoxazine – synthesis,spectroscopic properties,and potential application as sensors for l-cysteine

Several benzo[a]phenoxazine dyes containing a maleimide moiety have been synthesised and characterised by proton nuclear magnetic resonance spectroscopy and mass spectrometry. The spectroscopic properties, such as absorption and emission spectra, of these dyes – 9-maleimido-5H-benzo[a]phenoxazin-5-one ( 7a ), 6-chloro-9-maleimido-5H-benzo[a]phenoxazin-5-one ( 7b ), and 6-bromo-9-maleimido-5H-benzo[a]phenoxazin-5-one ( 7c ) – were examined. These compounds were evaluated as potential fluorescent and colorimetric probes for the detection of biothiols. Results show that the studied compounds exhibit colorimetric and fluorescent responses to l -cysteine at pH 7.4. In the presence of l -cysteine, the fluorescence intensity of synthesised dyes is greatly enhanced. The addition of l -cysteine to solutions of dyes 7a to 7c also results in red shifts of their absorption bands. The cytotoxic effect of tested dyes 7a to 7c against human neuroblastoma cells (SH-SY5Y) was determined by MTT assay. The fluorescent response of these compounds to thiols SH-SY5Y was examined. The results show that the tested compounds exhibit fluorescent response in this cell line, and the benzo[a]phenoxazine dyes substituted in the 6-position with a halogen ( 7b and 7c ) are less toxic against cells under experimental conditions than their unsubstituted analogue ( 7a ).     

Optimized fluorescent trimethoprim derivatives for in vivo protein labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.     

Detecting Cysteine in Bioimaging with a Near-Infrared Probe Based on a Novel Fluorescence Quenching Mechanism

Near-infrared (NIR) fluorescent probes are very significant for detecting cysteine in biological systems. Herein, we report a highly selective and sensitive NIR turn-on fluorescent probe (BDP-NIR) based on BODIPY with large Stokes shift (105 nm) for detecting Cys. We clarified the sensing mechanism based on the different thiol-induced S_NAr substitution/rearrangement reaction of the probe with cysteine and homocysteine/glutathione, which leads to the corresponding amino- and thiol-BODIPY dyes with distinct photophysical properties. Moreover, a novel mechanism of fluorescence quenching was demonstrated by density functional theory calculation. The reason for the fluorescence quenching of the probe might be intersystem crossing (from singlet to triplet excited state). Moreover, BDP-NIR had a high linear dynamic range of 0–500 μM, which was promising for detecting cysteine quantificationally. Significantly, BDP-NIR was capable of sensing endogenous cysteine in living cells and in vivo.     

Fluorochrome and Fluorescent In Situ Hybridization to Monitor Bioaerosols in Swine Buildings

Total microbial cell concentration, viability, and culturability of bioaerosols in swine buildings were monitored by using epifluorescence microscopy with fluorochrome (EFM/FL) with four fluorescent dyes (AO, DAPI, PI, and YOPRO-1) and by using fluorescent in situ hybridization (FISH) with five oligonucleotide probes (fl-Univ, fl-EUB, cy-EUK, fl-PSMg, and fl-NotEUB) probes. Results from these two non-culture-based methods were then compared with those using a commonly used culture method. The total microbial cell concentration measured using the non-culture-based methods was 10 to 200 times higher than that using the culture method; from 5.48 × 10⁶ to 2.18 × 10⁷ cells/m³ with AO staining and from 5.03 × 10⁶ to 2.13 × 10⁷ cells/m³ with DAPI staining, compared with the average concentration of 1.02 × 10⁵ CFU/m³ for bacteria and 1.27 × 10³ CFU/m³ for fungi by the culture method. The viability ranged from 0.27 to 0.76 by EFM/FL with PI staining, from 0.02 to 0.60 with YOPRO-1 staining, from 0.53 to 0.79 by FISH, and from 0.002 to 0.033 by the culture method. The viability by EFM/FL and FISH were much higher than the culturability. In summary, the total microbial cell concentration and viability were highly underestimated by the culture method. Based on the FISH results, eubacteria and eukaryotes were the dominant components of the bioaerosols. In conclusion, EFM/FL and FISH methods can effectively assess the total microbial cell concentration and viability of bioaerosols in environmental samples.     

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.     

Harnessing Fluorescent Moenomycin A Antibiotics for Bacterial Cell Wall Imaging Studies

The imaging of peptidoglycan (PGN) dynamics in living bacteria facilitates the understanding of PGN biosynthesis and wall-targeting antibiotics. The main tools for imaging bacterial PGN are fluorescent probes, such as the well-known PGN metabolic labeling probes. However, fluorescent small-molecule probes for labeling key PGN-synthesizing enzymes, especially for transglycosylases (TGases), remain to be explored. In this work, the first imaging probe for labeling TGase in bacterial cell wall studies is reported. We synthesized various fluorescent MoeA-based molecules by derivatizing the natural antibiotic moenomycin A (MoeA), and used them to label TGases in living bacteria, monitor bacterial growth and division cycles by time-lapse imaging, and study cell wall growth in the mecA-carrying methicillin-resistant Staphylococcus aureus (MRSA) strains when the β-lactam-based probes were unsuitable.     

Application of Neuron-Selective Fluorescent Probe,NeuA, To Identify Mouse Retinal Degeneration

The retina is part of the central nerve system (CNS) and has various interneurons and sensory neurons such as photoreceptor cells. Retinitis pigmentosa (RP) is an inherited condition that is characterized by photoreceptor degeneration. Herein, we developed a fluorescent probe-NeuA-for detecting retinal neuronal cells and applied NeuA to discriminate between healthy and RP retinas. The staining pattern of NeuA in the retinas of healthy and RP mouse models was examined in vitro, ex vivo and in vivo using confocal microscopy, the fluorescent fundus microscopy and optical coherent tomography (OCT). NeuA strongly stained the outer segment layer of photoreceptor cells and some bipolar cells in the healthy retina, but there was only weak staining in the photoreceptor degenerated retinas. Therefore, NeuA probe can be used as the detecting RP tools in the preclinical conditions.     

Design,Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for their binding affinity to the 18-kDa translocator protein (TSPO). Spectroscopic studies including UV/Vis absorption and fluorescence measurements showed that the synthesized fluorescent probes exhibit favorable spectroscopic properties, especially in nonpolar environments. In vitro fluorescence staining in brain sections from lipopolysaccharide (LPS)-injected mice revealed partial colocalization of the probes with the TSPO. The TSPO binding affinity of the probes was measured on crude mitochondrial fractions separated from rat brain homogenates in a [¹¹C]PK11195 radioligand binding assay. All the new fluorescent probes demonstrated moderate to high binding affinity to the TSPO, with affinity (K_i) values ranging from 0.58 nM to 3.28 μM. Taking these data together, we propose that the new fluorescent probes could be used to visualize the TSPO.     

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.     

Surface modification with polyallylamines for adhesion of biopolymers and cells

Cationic polymers with NH₂-groups were used for modification of charged and uncharged surfaces of planar slides, wells of plates, and spherical nanoparticles. Our study was aimed at development of a simple functionalization method of plain surfaces and colloids of different chemical compositions for adhesion of native biopolymers, including proteins, and viable bacterial and eukaryotic cells. Poly(allylamine)s (pAA) and polylysines (pLys) of different molecular weights spontaneously formed interfaces convenient for adhesion of biopolymers and cells. Thickness of the pAA 65 kDa layer ∼1.5–2 nm was measured by two methods: 1) atomic force microscopy (AFM) on mica slides and 2) registration of the long range surface optical waves excitation angle and the critical angle of total internal reflection from the liquid on a photonic crystal surface by using the biosensor. The sorption capacity of 0.1 mg/ml pAA 65 kDa exceeded the values of other polyamines at different concentrations. Physisorption of proteins on pAA layer was reversible and up to 70% of attached proteins could be removed by subsequent washes. Additional treatment with glutaraldehyde (GA) provided stable chemical cross-linking of the compounds containing primary NH₂-groups with aminated surfaces. The proteins immobilized on the pAA-covered surface retained their ability to bind with specific monoclonal and polyclonal antibodies. Bacterial cells after adhesion on pAA65-covered surfaces maintained their morphology, could reproduce and express the green fluorescent protein (gfp) gene under control of the inducible lac promoter. Eukaryotic cells of human and mammalian origin also remained viable on pAA-treated slides as proven by their staining with fluorescent dyes and cell divisions until confluent monolayers. Mammalian cells could not attach onto silicon wafers but grew on pAA interface of the silicon slides until confluent monolayers. Thus, surface modification with polyallylamines provides adhesion of native biopolymers and living cells.     

A Luminescent Cyclometalated Iridium(III) Complex Accumulates in Mitochondria and Induces Mitochondrial Shortening by Conjugation to Specific Protein Targets

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)₂(phen‐ITC)](PF₆) (Ir‐ITC; Hpq=2‐phenylquinoline, phen‐ITC=5‐isothiocyanate‐1,10‐phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir‐ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir‐ITC with live‐cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two‐dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage‐dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir‐ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.     

水溶性吲哚菁染料的合成、光谱性能及其应用的研究

设计、合成了3种水溶性吲哚菁染料（发射波长：690 nm）,用核磁共振（NMR）对其进行了表征,测试了菁染料在不同溶剂中的紫外吸收光谱和荧光发射光谱;为了探讨菁染料在生物分析领域的应用性,测定3种菁染料在十六烷基三甲基溴化铵（CTAB）和十二烷基磺酸钠（SDS）两种表面活性剂胶束模拟的生理条件下的吸收和发射光谱,利用循环伏安法（CV）测定了菁染料的光稳定性,在近红外区域观察了染料对肾癌细胞的活细胞染色性能,并计算了3种菁染料的摩尔吸光系数和荧光量子产率。     

HER2‐ and EGFR‐Specific Affiprobes: Novel Recombinant Optical Probes for Cell Imaging

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell‐surface receptors/tyrosine kinases. EGFR‐ and HER2‐positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR‐ or HER2‐negative cancers. The details of HER2 proto‐oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009 , 315, 683–696). We have created photostable and relatively simple‐to‐produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2‐ or EGFR‐specific Affibody® molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell‐surface antigens/ receptors.     

Modular Site-Specific Conjugation of Nanobodies Using Two Co-Associating Tags

The homogeneous labeling of antibodies and their fragments is a critical step for the generation of robust probes used in immuno-detection applications. To date, numerous chemical, genetic and peptide-based site-specific coupling methods have been developed. Among these methods, co-assembling peptide-tags is one of the most straightforward and versatile solutions. Here, we describe site-specific labeling of nanobodies through the use of two co-associating peptides tags, E3 and K3, originating from the tetramerization domain of p53. These E3 and K3-tags provide a simple and robust method for associating stoichiometric amount of V_HH and fluorescent probes, either fluorescent proteins or fluorochromes, at specific positions. As a proof of concept, a nanobody targeting the human epidermal growth factor receptor 2 (HER2), the nano-HER2 was genetically fused to the E3 and associated with different fluorescent K3-derivates. Entities were produced separately in Escherichia coli in soluble forms at high yields and co-assembled in vitro. These molecular probes present high binding specificity on HER2-overexpressing cells in flow-cytometry with relative binding constants in the low nanomolar range and are stable enough to stain HER2-receptor on living cells followed detection using fluorescent confocal microscopy. Altogether, our results demonstrate that the non-covalent conjugation method using these two co-associating peptides can be easily implemented for the modular engineering of molecular probes for cell immuno-staining.     

Synthesis and spectroscopic investigation of cationic donor‐(π‐bridge)‐acceptor dye, 4‐[N‐(5,6,7,8‐tetrahydroisoquinolinium‐5‐ylidene)methyl]‐N,N‐dialkylaniline iodide

The synthesis and fundamental spectroscopic properties of eight hemicyanine (HC) dyes are presented. The dyes were prepared by the condensation of N‐methyl‐5,6,7,8‐tetrahydroisoquinolinium iodide with p‐(N,N‐dialkylamino)benzaldehydes. The compounds were characterised by nuclear magnetic resonance spectroscopy and their purity was checked with the use of thin‐layer chromatography. The spectroscopic properties of the dyes were determined in three organic solvents. The electronic absorption spectra of the dyes demonstrate moderate sensitivity to the nature of the substituent present in the aromatic ring and low solvent polarity dependence. In contrast to this, the positions of fluorescence bands are affected by the structure of an electron donor and solvent polarity. The 4‐[N‐(5,6,7,8‐tetrahydroisoquinolinium‐5‐ylidene)methyl]‐N,N‐dialkylaniline iodides were applied as fluorescent probes for the monitoring of the progress of free radical polymerisation. The study on the changes in the fluorescence intensity and spectroscopic shifts of the dyes was carried out during thermally initiated polymeriszation of methyl methacrylate. The purpose of these studies was to find a relationship between the changes in the shape and intensity of probe fluorescence and the degree of monomer conversion into polymer.     

Bright fluorescent dsDNA probes: novel polycationic asymmetric monomethine cyanine dyes based on thiazolopyridine‐quinolinium chromophore

Seven tri‐ and tetracationic monomeric and homodimeric monomethine cyanine dyes based on thiazolo[4,5‐b]pyridinium and quinolinium end groups were synthesised and characterised. The dyes were tested as fluorescent DNA intercalating probes to apply in DNA gel electrophoresis. The DNA samples stained with all dyes from the series demonstrated bright fluorescent signals. DNA fragments were successfully visualised under orange and green filters as well as under standard UV transillumination. Two of the studied dyes revealed higher sensitivity to DNA when compared with the commercial dimeric cyanine dye TOTO‐1. Their sensitivity reached that of the commercial dimeric cyanine dye YOYO‐1, but the emission was shifted to longer wavelengths. These qualities make the dyes suitable to apply in a wide range of medical and scientific analytical methods.     

Novel,N-ethyl-2-styrylquinolinum iodides as fluorophores for monitoring of polymerization process,Part I

A series of of p-substituted 2-styrylquinolinium iodides were prepared by the condensation of N-ethyl-2-methylquinolinium salts with p-substituted benzaldehydes. The spectroscopic properties of the styryl quinolinium dyes are characterized in organic solvents of varying polarities. The electronic absorption and fluorescence emission spectra of the dyes demonstrate their high sensitivity to the nature of substituents introduced into the aromatic ring. The dyes were investigated as fluorescent probes for monitoring the progress of the photochemically initiated free-radical polymerization of a mixture of 2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate and 1-methyl-2-pyrrolidinone. During the course of the polymerization an increase in the fluorescence intensity of the dyes by at least one order of magnitude was recorded; a feature which renders the dyes as good fluorescent probes for such polymerization reactions. The term “probe sensitivity” has been defined and appears in the range from 0.08 to 11 for the styryl dyes.     

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Classical fluorescence‐based approaches to monitor ligand–protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn‐on probes for a G protein‐coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor‐specific turn‐on response. The new ligand was successfully applied for background‐free imaging and quantification of oxytocin receptors in living cells.