Current Progress in Expansion Microscopy: Chemical Strategies and Applications.

Expansion microscopy (ExM) is a newly developed super-resolution technique, allowing visualization of biological targets at nanoscale resolution on conventional fluorescence microscopes. Since its introduction in 2015, many efforts have been dedicated to broaden its application range or increase the resolution that can be achieved. As a consequence, recent years have witnessed remarkable advances in ExM. This review summarizes recent progress in ExM, with the focus on the chemical aspects of the method, from chemistries for biomolecule grafting to polymer synthesis and the impact on biological analysis. The combination of ExM with other microscopy techniques, in search of additional resolution improvement, is also discussed. In addition, we compare pre- and postexpansion labeling strategies and discuss the impact of fixation methods on ultrastructure preservation. We conclude this review with a perspective on existing challenges and future directions. We believe that this review will provide a comprehensive understanding of ExM and facilitate its usage and further development.

Improved Dye Survival in Expansion Microscopy through Stabilizer-Conjugated Linkers.

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer-containing organic fluorophores in ExM, as a mitigation strategy for this radical-induced dye degradation. Through direct conjugation of 4-nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore-conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer-modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.

Design, synthesis, DFT calculations, molecular docking and antimicrobial activities of novel cobalt, chromium metal complexes of heterocyclic moiety-based 1,3,4-oxadiazole derivatives.

A Schiff base, 5-(4-methylphenyl)-4-[(pyridin-2-ylmethylidene)amino]-4H-1,2,4-oxadiazole as a bidentate ligand has been synthesized by the reaction between the 4-amino-5-(4-methylphenyl)-4H-1,3,4-oxadiazole and aromatic aldehyde. The Schiff base reacted with CoCl3·6H2O and CrCl3·6H2O in ethanol to yield 1,3,4-oxadiazole complexes. The structures of synthesized ligand and their complexes have been established on the basis of their IR, Mass and 1H-NMR spectra. Electronic and geometric structures of both cobalt and chromium complexes were investigated by density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) calculations. DFT-based reactivity calculations estimated the studied system as strong electrophile and/or strong nucleophile in polar organic reactions. Moreover, most reactive sites were predicted theoretically based on the delocalized and localized indexes. The nature of Ligand-Metal chemical bonding is discussed in terms of the natural bond orbital (NBO) and QTAIM analysis. Accordingly, the metal ions such as cobalt and chromium are bidentate coordinated with the Schiff base by nitrogen atoms of imine function and pyridine, to form stable complexes. Furthermore, the chromium ions have an affinity superior to the cobalt ions towards Schiff base ligand. In addition, the results of the antibacterial activity in-vitro show that the metal complexation confers an increase in the antibacterial activity of the complexed ligand compared to the free ligand against both Gram-positive and Gram-negative bacteria with broad spectrum activity. In silico molecular docking studies of the ligands and their complexes were applied to describe the probable binding modes into the active site of Escherichia coli (E. coli) FabH and Salmonella typhimurium LT2 neuraminidase (STNA) receptors. The increase in biological activity could be attributed to the high stability of the complexes and strong affinities to bacterial enzyme receptors.Communicated by Ramaswamy H. Sarma.

Catalysis, Kinetic and Mechanistical Studies for the Transformation of Ethylene Glycol by Alumina and Silica Gel under Autogenous Pressure and Solvent-free Conditions.

A kinetic and mechanistical studies of the new pathway for competitive transformation of ethylene glycol by alumina and silica gel have been described. Commercial alumina (Al com), synthetic alumina (Al syn), commercial silica gel (Si com) and synthetic silica gel (Si syn) were used for the transformation of ethylene glycol to a mixture of diethylene glycol, 1,4-dioxane and 2-methyl-1,3-dioxolane via acetaldehyde by heating at 150 °C under autogenous pressure without solvent. The results show that the yield of these three products strongly depends on the nature of the used catalyst and the reaction time.

A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy.

Expansion microscopy (ExM) enables the nanoscale imaging of ribonucleic acids (RNAs) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents that allow the labeling and grafting of deoxyribonucleic acid (DNA) oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA FISH (fluorescence in situ hybridization) techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multicolor staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ∼50 nm in ×10 ExM for both pre- and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable the rapid functionalization of DNA oligonucleotides for ExM.

Synthesis, Characterization, and Antimicrobial Activity of a Novel Trisazo Dye from 3-Amino-4H-thieno[3,4-c][1]benzopyran-4-one.

A new trisazo dye has been synthesized by coupling the diazonium ion of 3-amino-4H thieno[3,4-c][1]benzopyran-4-one with 2-tert-butyl-4-methoxyphenol. The newly prepared trisazo dye was characterized by its physical, elemental, and spectroscopic data. 2D-NMR (COSY, HSQC, and HMBC) techniques were used to secure the structural assignments. The new trisazo dye (compound 7) along with precursors 3, 4, and 6 was screened by microdilution susceptibility assay for antibacterial and antifungal activities towards eight bacterial strains and three yeasts selected on the basis of their relevance as human pathogens. The results showed that compound 7 (MIC = 2-128 µg/mL) was the most active as compared with its precursors. The most resistant microorganisms were V. cholerae NB2 and V. cholerae SG24, whereas the most sensitive microorganism was C. neoformans. The overall results of this study indicated that compound 7 had the greatest potential value against both yeasts and multidrug-resistant bacteria, so further investigation is warranted.

Electrochemical behavior and in-vitro antimicrobial screening of some thienylazoaryls dyes.

BACKGROUND: A series of recently reported phenolic azo dyes 7a-e were prepared by coupling the thienyl diazonium sulfate of 3-Amino-4H-benzo[f]thieno[3,4-c](2H)chromen-4-one with selected diversely substituted phenolic and naphtholic derivatives. These compounds were evaluated for their antibacterial and antifungal activities. Furthermore their voltammetric behavior was compared at a glassy carbon electrode. RESULTS: The voltammetric behavior of the five recently reported azo dyes has been compared at a glassy carbon electrode. It is shown that the azo dyes 7a-e with a hydroxyl group in the ortho position with respect to the azo bridge give rise to well defined, irreversible peaks for the oxidation and reduction process within a pH range of 2-7. The mechanisms of electrochemical oxidation of compound 7a-c and 7e are proposed. For the hydroxyl-substituted dyes, re-oxidation peaks were obtained in the subsequent scan. The antimicrobial activities of the reported compounds 7a-e along with the entire precursors 1-4 and 6a-e were performed against selected bacterial and fungal species and their activities compared to those of nystatin, griseofulvin and ciprofloxacin used as reference drugs. CONCLUSIONS: The present study showed significant antimicrobial activity of compounds 6d, 7a and 7c,e against the tested microorganisms; this result confirms the antimicrobial potency of azo compounds and some of their precursors.

Synthesis, spectroscopy, crystal structure, electrochemistry, and quantum chemical and molecular dynamics calculations of a 3-anilino difluoroboron dipyrromethene dye.

An asymmetrically substituted fluorescent difluoroboron dipyrromethene (BODIPY) dye, with a phenylamino group at the 3-position of the BODIPY chromophore, has been synthesized by nucleophilic substitution of 3,5-dichloro-8-(4-tolyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. The solvent-dependent spectroscopic and photophysical properties have been investigated by means of UV-vis spectrophotometry and steady-state and time-resolved fluorometry and reflect the large effect of the anilino substituent on the fluorescence characteristics. The compound has a low fluorescence quantum yield in all but the apolar solvents cyclohexane, toluene, and chloroform. Its emission maxima in a series of solvents from cyclohexane to methanol are red-shifted by approximately 50 nm in comparison to classic BODIPY derivatives. Its oxidation potential in dichloromethane is at ca. 1.14 V versus Ag/AgCl. The absorption bandwidths and Stokes shifts are much larger than those of typical, symmetric difluoroboron dipyrromethene dyes. The values of the fluorescence rate constant are in the (1.4-1.7) x 10(8) s(-1) range and do not vary much between the solvents studied. X-ray diffraction analysis shows that the BODIPY core is planar. Molecular dynamics simulations show that there is no clear indication for aggregates in solution.

Electroactive dipyrromethene-Cu(II) self-assembled monolayers: complexation reaction on the surface of gold electrodes.

In the work presented, thiol- and COOH-terminated dipyrromethene derivatives have been applied for gold electrode modification. Dipyrromethene deposited onto a solid support, after binding Cu2+, can act as a redox active monolayer. The complexation of Cu(II) ions has been performed on the surface of gold electrodes modified with dipyrromethene. The characterization of dipyrromethene-Cu(II) self-assembled monolayers (SAMs) has been done by cyclic voltammetry (CV), wettability contact angle measurements, and atomic force microscopy (AFM). The new electroactive monolayer could be applied for the immobilization of proteins and ssDNA or for electrochemical anion sensing without redox markers in the solution.

Photophysics of 3,5-diphenoxy substituted BODIPY dyes in solution.

We have prepared two fluorescent dyes derived from 8-(4-tolyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene with phenoxy and (o-bromo)phenoxy substituents at the 3,5-positions by a novel nucleophilic substitution reaction of the corresponding 3,5-dichloroBODIPY analogue. UV-vis absorption, steady-state and time-resolved fluorimetry have been used to investigate their solvent-dependent photophysical properties. The two BODIPY derivatives show narrow absorption and emission bands and display small Stokes shifts. The substituents at the 3,5-positions (phenoxy in 1 and o-bromophenoxy in 2) have a minor effect on the fluorescence quantum yields (0.16-0.40 for 1, 0.17-0.44 for 2) and lifetimes (1.09-2.51 ns for 1, 1.11-2.78 ns for 2). For both compounds, the fluorescence rate constant equals (1.5 +/- 0.1) x 10(8) s(-1).

Boron dipyrromethene analogs with phenyl, styryl, and ethynylphenyl substituents: synthesis, photophysics, electrochemistry, and quantum-chemical calculations.

Seven fluorescent boradiazaindacene-based compounds with one or two phenyl, ethenylphenyl, and ethynylphenyl substituents at the 3- (or 3,5-) position(s) were synthesized via palladium-catalyzed coupling reactions with the appropriate 3,5-dichloroBODIPY derivative. The effect of the various substituents at the 3- (or 3,5-) position(s) on the spectroscopic and photophysical properties were studied as a function of solvent by means of UV/vis absorption, steady-state, and time-resolved fluorometry, and theoretical modeling. The emission maxima of the symmetrically 3,5-disubstituted dyes are shifted to longer wavelengths (by 30 to 60 nm) relative to the related asymmetrically 3,5-disubstituted ones. Introduction of styryl substituents causes the largest red shift in both the absorption and emission spectra. BODIPY derivatives with ethynylaryl groups also shift the spectral maxima to longer wavelengths compared to aryl-substituted ones but to a lesser degree than the styryl compounds. The quantum-chemical calculations confirm these trends and provide a rationale for the spectral shifts induced by substitution. The fluorescence quantum yields of the ethenylaryl and ethynylaryl analogs are significantly higher that those of the aryl-substituted dyes. The 3,5-diethynylaryl dye has the highest fluorescence quantum yield (approximately 1.0) and longest lifetime (around 6.5 ns) among the BODIPY dyes studied. The differences in the photophysical properties of the dyes are also reflected in their electrochemical properties where the symmetrically 3,5-disubstituted dyes display much lower oxidation potentials when compared to their asymmetric counterparts.

Functionalisation of fluorescent BODIPY dyes by nucleophilic substitution.

The BODIPY chromophore can be easily modified by nucleophilic mono- or disubstitution of 3,5-dichloroBODIPY with O-, N-, S- and C-nucleophiles. Absorption and fluorescence spectral data of the new BODIPY derivatives are also reported.

A highly potassium-selective ratiometric fluorescent indicator based on BODIPY azacrown ether excitable with visible light.

[structure: see text] A potassium-selective fluorescent BODIPY-linked azacrown ether chemosensor has been synthesized using novel substitution reactions of 3,5-dichloroBODIPY. The indicator absorbs and emits light in the visible wavelength range. The dissociation constant Kd for the complex between K+ and the probe is 0.5 mM in acetonitrile. Quantum chemical calculations show that the experimental hypsochromic shifts in absorption and fluorescence upon potassium binding are due to complexation, which induces a large conformational change of the sensor.