Epoxy-amine Reaction Research Articles

A comparison study of graphene-cyclodextrin conjugates for enhanced electrochemical performance of tyramine compounds

The present work describes the comparison of the abilities of graphene-cyclodextrin conjugates to enhance the electrochemical performance of four tyramine-related compounds. First, cyclodextrin (CD)-modified graphene conjugates were synthesized via the amine-epoxy reaction between graphene oxide (GO) and 6-deoxy-6-ethylenediamino-β-CD, followed by l-ascorbic acid reduction. The resulting conjugates were characterized using UV–vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analysis (TGA), X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Subsequently, for the comparative study, glass carbon electrode electrochemical sensors modified with these conjugates were prepared to detect four structurally similar analytes (tyramine, l-tyrosine, dopamine and levodopa). The sensor sensitivities of the modified electrodes were markedly higher than that of the bare electrode. Based on the results of the comparative study, several crucial factors for improving the performance of the electrodes were proposed, including the binding affinity and the binding orientation of CD toward analytes. The conclusions drawn in this study could provide theoretical foundation for the design and optimization of versatile electrochemical platforms with excellent properties.

In situ hyper-cross-linking of glycidyl methacrylate–based polyHIPEs through the amine-enriched high internal phase emulsions

The synthesis of glycidyl methacrylate–based polyHIPEs by free-radical polymerization in the presence of 1,8-diaminoctane or tris(2-aminoethyl)amine-enriched high internal phase emulsions is presented. This innovative “one-pot” synthetic route was developed to produce the so-called in situ hyper-cross-linked polyHIPEs without the use of any additional catalyst, cross-linker, or solvent. In situ hyper-cross-linking was performed through the amine-epoxy reaction before the gel point had been reached, resulting in the formation of the β-amino alcohol derivatives that represent cross-linking knots between the neighboring epoxy repeating units within the poly (glycidyl methacrylate) backbone. In this way, the volume of the mesopores smaller than 3 nm significantly increased. Thus, by changing the amounts of ethylene glycol dimethacrylate and amines in the HIPE templates, the porous structure and the pore volume of the hyper-cross-linked polyHIPEs were systematically altered in order to amplify the polyHIPE’s specific surface area.

Cyclophosphazene-based hybrid polymer electrolytes obtained via epoxy–amine reaction for high-performance all-solid-state lithium-ion batteries

This article reports a cyclophosphazene-based hybrid polymer electrolyte formed via the epoxy–amine reaction for high-performance lithium-ion batteries.

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A reversed phase (RP)/hydrophilic interaction (HILIC)/ion exchange (IEX) mixed tri-mode stationary phase (TMSP) has been prepared via a divergent synthesis scheme starting from propylamine on silica then by amine-epoxy reactions with 1,4-butanedioldiglycidyl ether and tertiary amines (N,N-dimethyldecylamine, DMDA). Its retention mechanism was found to follow RP/HILIC/IEX mixed-mode. The stop-flow test revealed that TMSP had good compatibility with 100% aqueous mobile phase. It demonstrated effective separation towards several kinds of compounds or drug molecules and their counterions within a single run.

Thickness effect on the generation of temperature and curing degree gradients in epoxy–amine thermoset systems

A thermo-kinetic model was employed to study the temperature and curing degree distribution in a casting part of a DGEBA–DDM system during its curing process in an oven. Initially, the curing of the DGEBA–DDM casting part system was investigated by isothermal and non-isothermal differential scanning calorimetry. A Kamal and Sourour phenomenological model expanded by a diffusion factor was proposed for modeling the curing. The proposed model fit properly the curing behavior of this system in the analyzed range of temperatures. This model enables the application within finite element analysis software for modeling the curing process of real thermosetting parts. Finite element-based program COMSOL Multiphysics™ was used to simulate the curing process. The model fits properly the initial heating of the sample until the reaction temperature, the time position at which the temperature starts to increase due to the heat generated during epoxy–amine reaction and also the rate at which the temperature increases, but it overestimates the maximum temperatures reached in the system. Nevertheless, the proposed model is shown as a powerful tool to design optimal curing cycles for thermosetting resins avoiding temperatures closer to the degradation temperature of the system and avoiding significant temperature gradients inside the sample.

Nanocomposites based on graphene oxide and mesoporous silica nanoparticles: Preparation, characterization and nanobiointeractions with red blood cells and human plasma proteins

The current work refers to the development of a novel nanocomposite based on graphene oxide (GO) and mesoporous amino silica nanoparticles (H2N-MSNs) and its biological interaction with red blood cells (RBCs) and human blood plasma toward the investigation of nanobiointeractions. Silica nanoparticles and several graphene oxide-based materials are, separately, known for their high hemolytic potential and strong interaction with human plasma proteins. In this context, the GO-MSN interaction and its influence in minimizing the reported effects were investigated. The materials were synthesized by covalently attaching H2N-MSNs onto the surface of GO through an amidation reaction. GO-MSN nanocomposites were obtained by varying the mass of H2N-MSNs and were characterized by FTIR, NMR, XRD, TGA, zeta potential and TEM. The characterization results confirm that nanocomposites were obtained, suggest covalent bond attachment mostly by amine-epoxy reactions and evidence an unexpected reduction reaction of GO by H2N-MSNs, whose mechanism is proposed. Biological assays showed a decrease of hemolysis (RBC lysis) and a minimization of the interaction with human plasma proteins (protein corona formation). These are important findings toward achieving in vivo biocompatibility and understanding the nanobiointeractions. Finally, this work opens possibilities for new nanomedicine applications of GO-MSN nanocomposites, such as drug delivery system.

Preparation of a reversed-phase/anion-exchange mixed-mode spherical sorbent by Pickering emulsion polymerization for highly selective solid-phase extraction of acidic pharmaceuticals from wastewater

The present work represents a simple and effective preparation of a novel mixed-mode anion-exchange (MAX) sorbent based on porous poly[2-(diethylamino)ethyl methacrylate-divinylbenzene] (poly(DEAEMA-DVB)) spherical particles synthesized by one-step Pickering emulsion polymerization. The poly(DEAEMA-DVB) particles were quaternized with 1,4-butanediol diglycidyl ether (BDDE) followed by triethylamine (TEA) via epoxy-amine reaction to offer strong anion exchange properties. The synthesized MAX sorbent was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, nitrogen adsorption-desorption measurements and elemental analysis. The MAX sorbent possessed regular spherical shape and narrow diameter distribution (15–35μm), a high IEC of 0.54meq/g, with carbon and nitrogen contents of 80.3% and 1.62%, respectively. Compared to poly(DEAEMA-DVB), the MAX sorbent exhibited decreased SBET (390.5 vs. 515.3m2g−1), pore volume (0.74 vs. 0.85cm3g−1) and pore size (16.8 vs. 17.3nm). Moreover, changes of N content for producing the MAX sorbent reveal a successful two-step quaternization, which can be highly related to such a high IEC. Finally, the MAX sorbent was successfully evaluated for selective isolation and purification of some selected acidic pharmaceuticals (ketoprofen, KEP; naproxen, NAP; and ibuprofen, IBP) from neutral (hydrocortisone, HYC), basic (carbamazepine, CAZ; amitriptyline, AMT) pharmaceuticals and other interferences in water samples using solid phase extraction (SPE). An efficient analytical method based on the MAX-based mixed-mode SPE coupled with HPLC-UV was developed for highly selective extraction and cleanup of acidic KEP, NAP and IBP in spiked wastewater samples. The developed method exhibited good sensitivity (0.009–0.085μgL−1 limit of detection), satisfactory recoveries (82.1%–105.5%) and repeatabilities (relative standard deviation < 7.9%, n=3).

Design of Carbon Dots Photoluminescence through Organo-Functional Silane Grafting for Solid-State Emitting Devices

Advanced optical applications of fluorescent carbon dots (C-dots) require highly integrated host-guest solid-state materials with a careful design of C-dots – matrix interface to control the optical response. We have developed a new synthesis based on the grafting of an organo-functional silane (3-glycidyloxypropyltrimethoxysilane, GPTMS) on amino-functionalized C-dots, which enables the fabrication of highly fluorescent organosilica-based hybrid organic-inorganic films through sol-gel process. The GPTMS grafting onto C-dots has been achieved via an epoxy–amine reaction under controlled conditions. Besides providing an efficient strategy to embed C-dots into a hybrid solid-state material, the modification of C-dots surface by GPTMS allows tuning their photoluminescence properties and gives rise to an additional, intense emission around 490 nm. Photoluminescence spectra reveal an interaction between C-dots surface and the polymeric chains which are locally formed by GPTMS polymerization. The present method is a step forward to the development of a surface modification technology aimed at controlling C-dots host-guest systems at the nanoscale.

Breathable Microgel Colloidosome: Gas-Switchable Microcapsules with O2 and CO2 Tunable Shell Permeability for Hierarchical Size-Selective Control Release.

Microcapsules enabling precise delivery and controlled release are highly desirable. However, it is still challenging to control the release profile by regulating the microcapsule shell permeability. In this work, gas-switchable microgel colloidosome (MGC) with oxygen (O2) and carbon dioxide (CO2) dual gas-tunable shell permeability has been developed and tested for control release of water-soluble cargo molecules, based on the size exclusion mechanism. The O2 and CO2 dual gas-switchable poly(2-(diethylamino)ethyl methacrylate-co-2,3,4,5,6-pentafluorostyrene), P(DEA-co-FS), microgels having surface modified with amino group (-NH2) were synthesized and used to stabilize oil-in-water (O/W) Pickering emulsions. The oil-soluble poly(propylene glycol) diglycidyl ether (PPGDGE) was added as an intermicrogel cross-linker. The cross-linking between adjacent microgel particles at the water-oil interface was achieved through the amine-epoxy reaction of PPGDGE with the amine groups at the particle surface. Fluorescent-labeled dextran model cargo molecules of 10 kDa (D1) and 2000 kDa (D2) were uploaded under CO2 treatment and locked inside the MGC with N2 treatment. The O2 and CO2 dual-gas switchable properties offered the MGC with tunable shell permeability, which allowed the hierarchical release of D1 and D2 based on size exclusive mechanism. This work provides a robust method for preparation of gas-switchable microcapsules with tunable permeability and size-exclusive hierarchical release profile, promising for multiple ingredient controllable release, separation, and reaction.

Durable superhydrophobic and antimicrobial cotton fabrics prepared by electrostatic assembly of polyhexamethylene biguanide and subsequent hydrophobization

Growing evidence shows that healthcare textiles act as reservoirs of pathogens responsible for healthcare-associated infections. Fabrics fortified with antimicrobial and superhydrophobic properties are slowly emerging as an ideal weapon to tackle these infections, because of their bactericidal and fluid-repellent functionalities. In this work, such dual functional fabrics were developed by depositing polyhexamethylene biguanide on cotton fabrics, followed by inclusion of an epoxy alkane/epoxy cross-linker. A layer-by-layer technique was employed for the incorporation of polyhexamethylene biguanide in place of the conventional single layer ionic (carboxylate anions of cellulose and cationic polyhexamethylene biguanide) interaction. The role of the epoxy cross-linker is paramount as it achieves cross-linking of polyhexamethylene biguanide chains and also ensures the anchoring of epoxyalkane to polyhexamethylene biguanide chains through amine–epoxy reaction. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the presence of polyhexamethylene biguanide and epoxyhexadecane on the fabric surfaces. The fabric surfaces exhibited high static water contact angles (&gt;150°) and lower water shedding angle (&lt;20°). The fabrics demonstrated impressive antimicrobial performance against Escherichia coli bacterial species. Importantly, in a separate protocol, the fabrics also decreased attachment of Escherichia coli cells by 70%, thus confirming their potential in the prevention of biofilm formation. Both the antimicrobial property and superhydrophobicity were retained after 50 equivalent home laundering cycles.

Versatile Bottom-Up Synthesis of Tethered Bilayer Lipid Membranes on Nanoelectronic Biosensor Devices.

Interfacing nanoelectronic devices with cell membranes can enable multiplexed detection of fundamental biological processes (such as signal transduction, electrophysiology, and import/export control) even down to the single ion channel level, which can lead to a variety of applications in pharmacology and clinical diagnosis. Therefore, it is necessary to understand and control the chemical and electrical interface between the device and the lipid bilayer membrane. Here, we develop a simple bottom-up approach to assemble tethered bilayer lipid membranes (tBLMs) on silicon wafers and glass slides, using a covalent tether attachment chemistry based on silane functionalization, followed by step-by-step stacking of two other functional molecular building blocks (oligo-poly(ethylene glycol) (PEG) and lipid). A standard vesicle fusion process was used to complete the bilayer formation. The monolayer synthetic scheme includes three well-established chemical reactions: self-assembly, epoxy-amine reaction, and EDC/NHS cross-linking reaction. All three reactions are facile and simple and can be easily implemented in many research labs, on the basis of common, commercially available precursors using mild reaction conditions. The oligo-PEG acts as the hydrophilic spacer, a key role in the formation of a homogeneous bilayer membrane. To explore the broad applicability of this approach, we have further demonstrated the formation of tBLMs on three common classes of (nano)electronic biosensor devices: indium-tin oxide-coated glass, silicon nanoribbon devices, and high-density single-walled carbon nanotubes (SWNT) networks on glass. More importantly, we incorporated alemethicin into tBLMs and realized the real-time recording of single ion channel activity with high sensitivity and high temporal resolution using the tBLMs/SWNT network transistor hybrid platform. This approach can provide a covalently bonded lipid coating on the oxide layer of nanoelectronic devices, which will enable a variety of applications in the emerging field of nanoelectronic interfaces to electrophysiology.

A Robust Cross-Linking Strategy for Block Copolymer Worms Prepared via Polymerization-Induced Self-Assembly.

A poly(glycerol monomethacrylate) (PGMA) chain transfer agent is chain-extended by reversible addition–fragmentation chain transfer (RAFT) statistical copolymerization of 2-hydroxypropyl methacrylate (HPMA) with glycidyl methacrylate (GlyMA) in concentrated aqueous solution via polymerization-induced self-assembly (PISA). A series of five free-standing worm gels is prepared by fixing the overall degree of polymerization of the core-forming block at 144 while varying its GlyMA content from 0 to 20 mol %. 1H NMR kinetics indicated that GlyMA is consumed much faster than HPMA, producing a GlyMA-rich sequence close to the PGMA stabilizer block. Temperature-dependent oscillatory rheological studies indicate that increasing the GlyMA content leads to progressively less thermoresponsive worm gels, with no degelation on cooling being observed for worms containing 20 mol % GlyMA. The epoxy groups in the GlyMA residues can be ring-opened using 3-aminopropyltriethoxysilane (APTES) in order to prepare core cross-linked worms via hydrolysis-condensation with the siloxane groups and/or hydroxyl groups on the HPMA residues. Perhaps surprisingly, 1H NMR analysis indicates that the epoxy–amine reaction and the intermolecular cross-linking occur on similar time scales. Cross-linking leads to stiffer worm gels that do not undergo degelation upon cooling. Dynamic light scattering studies and TEM analyses conducted on linear worms exposed to either methanol (a good solvent for both blocks) or anionic surfactant result in immediate worm dissociation. In contrast, cross-linked worms remain intact under such conditions, provided that the worm cores comprise at least 10 mol % GlyMA.

Poly ionic liquid cryogel of polyethyleneimine: Synthesis, characterization, and testing in absorption studies

ABSTRACTHere, we report the synthesis of polyethyleneimine (PEI) cryogels for the first time via cryopolymerization technique. The crosslinking of amine groups on the branched PEI chains is accomplished with epoxy groups of glycerol diglycidyl ether (GDE) based on epoxy–amine reactions in excess water at −18 °C in about 16 h. Superporous PEI cryogels with pore sizes &gt;100 μm were shown to have very fast equilibrium swelling behavior, e.g., 10 s to reach maximum swelling in DI water. Furthermore, the synthesized PEI cryogels were exposed to anion exchange reaction after protonation by HCl treatments to generate PEI ionic liquid cryogels containing hexafluorophosphate, thiocyanate, dicyanamide, and tetrafluoroborate. It was also demonstrated that PEI cryogels modified with [PF6]− absorbed 47.8 ± 5.7 mg/g of bovine serum albumin (BSA). Moreover, PEI cryogels were shown to be very useful as simple filtration filling materials for the direct removal of organic dyes such as methyl orange (MO) and eosin Y (EY) from their corresponding aqueous solutions with 98.5 and 98.6% yields, respectively. The separation of methylene blue (MB) from MO and EY mixture by using PEI cryogels as column filler materials was also demonstrated. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43478.

Glucaminium ionic liquid-functionalized stationary phase for the separation of nucleosides in hydrophilic interaction chromatography.

A glucaminium-based ionic liquid stationary phase was prepared via facile epoxy-amine reaction and subsequent quaternization. Successful immobilization of glucaminium-based ionic liquid onto silica surface was validated by elemental analysis and infrared spectroscopy. The new stationary phase was evaluated for the separation of nucleosides in hydrophilic interaction liquid chromatography (HILIC). Effects of various factors, such as acetonitrile concentration, salt concentration, pH value, as well as column temperature, on the chromatographic behavior toward nucleosides were studied in detail. The results indicated that this new stationary phase can be used for separation of water-soluble polar substances in HILIC mode. The retention of solutes on the stationary phase was influenced by a mixed-mode retention mechanism with a combination of adsorptive and partitioning interactions.

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics.

Non-isothermal cure and exfoliation of tri-functional epoxy-clay nanocomposites

The non-isothermal cure kinetics of polymer silicate layered nanocomposites based on a tri-functional epoxy resin has been investigated by differential scanning calorimetry. From an analysis of the kinetics as a function of the clay content, it can be concluded that the non-isothermal cure reaction can be considered to consist of four different processes: the reaction of epoxy groups with the diamine curing agent; an intra-gallery homopolymerisation reaction which occurs concurrently with the epoxy-amine reaction; and two extra-gallery homopolymerisation reactions, catalysed by the onium ion of the organically modified clay and by the tertiary amines resulting from the epoxy-amine reaction. The final nanos- tructure displays a similar quality of exfoliation as that observed for the isothermal cure of the same nanocomposite system. This implies that the intra-gallery reaction, which is responsible for the exfoliation, is not significantly inhibited by the extra-gallery epoxy-amine cross-linking reaction.

New redox-active layer create via epoxy–amine reaction – The base of genosensor for the detection of specific DNA and RNA sequences of avian influenza virus H5N1

This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a “click” reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal.

Improved copper corrosion resistance of epoxy-functionalized hybrid sol–gel monolayers by thiosemicarbazide

To improve the copper corrosion resistance of epoxy-functionalized hybrid sol–gel monolayers (Hy) consisting of 3-glycidoxypropyltrimethoxysilane (GPTMS-functional monomer) and tetraethoxysilane (TEOS-reticulating agent), the copper surface was modified by a well-defined inhibitor viz., thiosemicarbazide (TSC). At first, the copper surface was activated by TSC monolayers through Cu–S bonds which end up with free tail amino groups. Secondly, Hy monolayers were immobilized on the TSC-modified copper surface via epoxy-amine reaction. The interaction of TSC with copper and Hy was investigated by Fourier transform infrared spectroscopy, which revealed the cleavage of epoxy ring due to the cross-linking reaction with free amino groups of TSC monolayers. The surface morphology of these monolayers was investigated by scanning electron microscopy and atomic force microscopy. The effectiveness of TSC and Hy monolayers on corrosion protection of copper was scrutinized by electrochemical methods viz., potentiodynamic polarization studies and electrochemical impedance spectroscopy techniques. These studies revealed the enhancement of copper corrosion resistance of epoxy-functionalized Hy monolayers by the TSC activation.

Comparative DSC kinetics of the reaction of DGEBA with aromatic diamines IV. Iso-conversional kinetic analysis

Applying the differential scanning calorimetry, DSC, we discuss in this study the so called iso-conversional kinetics, ICK, of different epoxy–amine reactions. Single point model free methods, such as the method of Kissinger for the peak maximum temperature evolution in programmed temperature DSC regime, are applied in comparison. The dependencies of the activation energy versus degree of epoxy conversion are analyzed for the systems studied from the point of view of the probable reaction models. Important features and limitations of the ICK approach are also commented.

Multifunctional Photodegradable Polymers for Reactive Micropatterns

We report the first example of realizing the multifunctionalization of photodegradable polymers for the preparation of reactive micropatterns. Three o-nitrobenzaldehyde monomers (M1, M2, and M3) with allyl, propargyl, and epoxy groups were synthesized in high yields by the simple reactions of 5-hydroxy-2-nitrobenzaldehyde with allyl bromide, propargyl bromide, and epichlorohydrin, respectively. Passerini multicomponent polymerization (Passerini MCP) of M1, M2, or M3 with 1,6-hexanedioic acid and 1,6-diisocyanohexane generated three poly(ester–amide)s (P1, P2, and P3), the ester linkages of which were o-nitrobenzyl derivatives with functional groups from the corresponding monomers. Therefore, these polymers are photodegradable and can also be further modified by efficient click reactions like thiol–ene, copper catalyzed azide–alkyne cycloaddition (CuAAC), and epoxy–amine reaction. Furthermore, Passerini MCP of M1, M2, and M3 with 1,6-hexanedioic acid and 1,6-diisocyanohexane could yield a photodegradable triply functional polymer (P4) containing all the three functional side groups which can be further modified by sequential click reactions. All the polymers were thoroughly characterized by 1H NMR and GPC. Degradation of the polymers in solution under UV irradiation was investigated by UV–vis and GPC, and they can all be photocleaved into oligomers and small molecules in 30 min. These functional polymers are extremely useful as positive photoresists to create reactive micropatterns. As an example, the triply functional polymer film was fabricated and cross-linked by epoxy–amine reaction. After photoirradiation under a mask, reactive patterns with allyl and propargyl groups were obtained. Sequential modification of the reactive sites by CuAAC and thiol–ene reactions afforded multifunctional patterned surfaces with tunable properties as confirmed by scanning electron micrograph (SEM) and confocal fluorescence microscopy.