Hydrophilic Side Chains Research Articles

Rational design of acid-degradable polymeric micelles in response to root-mediated pH changes for site-specific herbicide delivery

The low utilization efficacy of conventional herbicide formulations greatly harms the environment and human health. Inspired by the fact that many drug delivery systems are designed to achieve targeted release triggered by the microenvironment of tumor tissues in cancer therapy, a tartaric acid-derived polyester with hydrophilic acid-degradable side chains was designed and synthesized for site-specific herbicide delivery by utilizing the acidic microenvironment in the rhizosphere. The amphiphilic polymer can spontaneously form polymeric micelles in the aqueous solution to load diuron. The diuron-loaded polymeric micelles can release diuron under acidic conditions, and the release rate increase with the reduction of pH value. The herbicidal effect of the diuron-loaded polymeric micelles is investigated using barnyard grass as the research object. The results reveal that the diuron-loaded polymeric micelles are able to kill barnyard grass effectively. Finally, the toxicity studies suggest that the polymer can mitigate toxicity to the non-target organisms. In view of its superior herbicidal activity, excellent biodegradability and biocompatibility, this site-specific herbicides release system has broad potential in agricultural applications.

Highly conductive and dimensionally stable anion exchange membranes enabled by rigid poly(arylene alkylene) backbones

Dimensionally and chemically stable anion exchange membranes with excellent ion conductivity are highly desired in electrochemical energy conversion technologies. Herein, we present a design using rigid arylene units, i.e., p-tetraphenylene and p-terphenylene, to suppress the water sorption of quaternized poly(arylene alkylene) membranes with high ion contents. Of note, side chain double-quaternary ammonium (QA) functionalized poly(q-tetraphenylene-co-p-terphenylene alkylene) membrane P(4PA-co-3PA)-D100 with the IEC value as high as 2.93 mmol g−1 showed very limited water uptake and swelling at 80 °C of 75 wt% and 14 %, respectively. Meanwhile, the immiscibility of hydrophilic double-cation side chain and the hydrophobic poly(arylene alkylene) backbone drives the formation of phase-separated morphology. As a result, P(4PA-co-3PA)-D100 showed an exceptional conductivity of 227.8 mS cm−1 at 80 °C with activation energy of 10.5 kJ mol−1. Despite the rigid poly(arylene alkylene) backbone with highly chemical durability and oxidative stability, the tandem tethered double cation in the side chain showed a 11 % QA moiety loss after a 1000-h treatment in 2 M NaOH at 80 °C. Moreover, a good peak power density of 550 mW cm−2 at the current density of 1.06 A cm−2 was achieved for P(4PA-co-3PA)-D100 with the gases flow of 200 mL min−1. This work demonstrates that, in addition to the approach of cross-linking, the highly conductive AEMs with excellent dimensional stability can be achieved by improving the rigidity of polymer backbones.

Perfluorosulfonic acid proton exchange membrane with double proton site side chain for high-performance fuel cells at low humidity

Structural optimization of ionomers is an effective strategy for achieving high-performance proton exchange membranes (PEMs) under low relative humidity (RH) conditions. In this study, sulfonimide group and trifluoromethanesulfonate acid (TFSA) ionic liquids were introduced to the perfluorosulfonic acid (PFSA) side chain, resulting in polymer membranes with varying chain lengths (i.e., PFC2-TF-SI, PFC4-TF-SI, and PFC5-TF-SI). This dual proton-conducting structure extended the length of the hydrophilic side chain and enhanced the hydrophobic-hydrophilic phase separation, aiding in the formation of proton transport channels. Notably, the proton conductivity of PFC5-TF-SI and PFC2-TF-SI membranes reached 7.1 and 10.6 mS/cm at 30% RH and 80 °C, respectively, which were approximately 29.1% and 92.7% higher than that of the pristine PFC5-SA membrane (5.5 mS/cm). Furthermore, the maximum power density of the PFC5-TF-SI and PFC2-TF-SI membranes from the built single fuel cell achieved 649 and 763 mW/cm2 at 30% RH and 80 °C, respectively, which were higher than that of the pristine PFC5-SA membrane (567 mW/cm2) by about 14.5% and 34.6%, respectively. Thus, this study provides a strategy for PEM design under low RH conditions.

Highly Efficient n‐Doping via Proton Abstraction of an Acceptor1‐Acceptor2Alternating Copolymer toward Thermoelectric Applications

AbstractElectron transporting (n‐type) polymers are the coveted complementary counterpart to more thoroughly studied hole transporting (p‐type) semiconducting polymers. Besides intrinsic stability issues of the doped form ofn‐type polymer toward ubiquitous oxidizing agents (H2O and O2), the choice of suitablen‐dopants and underlying mechanism of doping is an open research field. Using a low LUMO,n‐type unipolar acceptor1‐acceptor2copolymer poly(DPP‐TPD) in conjunction with bulkn‐doping using Cs2CO3these issues can be addressed. A solid‐state acid‐base interaction between polymer and basic carbonate increases the backbone electron density by deprotonation of the thiophene comonomer while forming bicarbonate, as revealed by NMR and optical spectroscopy. Comparable to N‐DMBI hydride/electron transfer, Cs2CO3proton abstraction doping shifts the poly(DPP‐TPD) work function toward the LUMO. Thereby, the anionic doped state is resilient against O2but is susceptible toward H2O. Based on GIWAXS, Cs2CO3is mostly incorporated into the amorphous regions of poly(DPP‐TPD) with the help of hydrophilic side chains and has minor impact on the short‐range order of the polymer. Cs2CO3proton abstraction doping and the acceptor1‐acceptor2copolymer architecture creates a synergisticn‐doped system with promising properties for thermoelectric energy conversion, as evidenced by a remarkable power factor of (5.59 ± 0.39) × µW m−1 K−2.

Nafion-like structured perfluoropoly(diphenylene) graft polymers microphase separated anion exchange membranes

Perfluorinated poly(diphenyl) graft polymers with alkoxy side chains of piperidinium quaternary ammonium salts were synthesized by two-step trifluoromethanesulfonic acid and lanthanum trifluoromethanesulfonate catalyzed and Williamson ether-forming reactions. Its grafted hydrophilic side chains can form a good microphase separation structure, as demonstrated by small angle X-ray scattering and atomic force microscope. The good phase separation structure improves the ionic conductivity while ensuring a smaller swelling ratio. The ionic conductivity of long side chain polymer (PF4) reached 80 mS cm−1 at 80 °C, while the swelling ratio was only 17.1 %. The maximum power density of PF4 was 1.008 W cm−2 at a gas flow rate of 200 sccm and a gas back pressure of 0.1 MPa. In addition, due to the perfluorinated substituted backbone and the non-N-site bonded piperidine cation, all the anion exchange membranes prepared in this paper possess good alkaline stability, with a conductivity drop of no more than 11 % after 1000 h of immersion in 4 M NaOH solution, and essentially no change in mechanical properties. The PF4 was operated at a current density of 200 mA cm−2 for 250 h with a voltage drop of only 97 mV and a resistance increase of 12 %.

Elucidating the Roles of Nafion/Solvent Formulations in Copper-Catalyzed CO2 Electrolysis.

Nafion ionomer, composed of hydrophobic perfluorocarbon backbones and hydrophilic sulfonic acid side chains, is the most widely used additive for preparing catalyst layers (CLs) for electrochemical CO2 reduction, but its impact on the performance of CO2 electrolysis remains poorly understood. Here, we systematically investigate the role of the catalyst ink formulation on CO2 electrolysis using commercial CuO nanoparticles as the model pre-catalyst. We find that the presence of Nafion is essential for achieving stable product distributions due to its ability to stabilize the catalyst morphology under reaction conditions. Moreover, the Nafion content and solvent composition (water/alcohol fraction) regulate the internal structure of Nafion coatings, as well as the catalyst morphology, thereby significantly impacting CO2 electrolysis performance, resulting in variations of C2+ product Faradaic efficiency (FE) by >3×, with C2+ FE ranging from 17 to 54% on carbon paper substrates. Using a combination of ellipsometry and in situ Raman spectroscopy during CO2 reduction, we find that such selectivity differences stem from changes to the local reaction microenvironment. In particular, the combination of high water/alcohol ratios and low Nafion fractions in the catalyst ink results in stable and favorable microenvironments, increasing the local CO2/H2O concentration ratio and promoting high CO surface coverage to facilitate C2+ production in long-term CO2 electrolysis. Therefore, this work provides insights into the critical role of Nafion binders and underlines the importance of optimizing Nafion/solvent formulations as a means of enhancing the performance of electrochemical CO2 reduction systems.

Side-Chain Engineering in Hydrophilic n-Type π-Conjugated Polymers for Enhanced Reactivity

Minor changes to side chains in conjugated polymers (CPs) can have pronounced effects on polymer properties by altering backbone planarity, solubility, and interaction with ions. Here, we report the photocontrolled synthesis of hydrophilic CPs from Grignard monomers and find that switching from alkyl to oligo(ethylene glycol) (OEG) side chains changes their photoreactivity. Specifically, installing hydrophilic side chains on the same monomer core yields higher molecular weight polymers and allows polymerization to proceed with lower-energy red light. Additionally, we discover a side chain decomposition pathway for N-OEG monomers, which are prevalent in CP research. Decomposition can be overcome by adding an extra methylene unit in the side chains without compromising polymer molecular weight or hydrophilicity. Importantly, this polymerization does not require transition metal catalysts and is a promising approach to the preparation of n-type conjugated block copolymers.

Ion Size-Dependent Electrochromism in Air-Stable Napthalenediimide-Based Conjugated Polymers.

Conjugated polymers (CPs) that show stable and reversible cation insertion/deinsertion under ambient conditions hold great potential for optoelectronic and energy storage devices. However, n-doped CPs are prone to parasitic reactions upon exposure to moisture or oxygen. This study reports a new family of napthalenediimide (NDI) based conjugated polymers capable of undergoing electrochemical n-type doping in ambient air. By functionalizing the NDI-NDI repeating unit with alternating triethylene glycol and octadecyl side chains, the polymer backbone shows stable electrochemical doping at ambient conditions. We systematically investigate the extent of volumetric doping involving monovalent cations of varying size (Li+, Na+, tetraethylammonium (TEA+)) with electrochemical methods, including cyclic voltammetry, differential pulse voltammetry, spectroelectrochemistry, and electrochemical impedance spectroscopy. We observed that introducing hydrophilic side chains on the polymer backbone improves the local dielectric environment of the backbones and lowers the energetic barrier for ion insertion. Surprisingly, when using Na+ electrolyte, the polymer films exhibit higher volumetric doping efficiency, faster-switching kinetics, higher optical contrast, and selective multielectrochromism when compared to Li+ or TEA+ electrolytes. Using well-tempered metadynamics, we characterize the free energetics of side chain-ion interactions to find that Li+ binds more tightly to the glycolated NDI moieties than Na+, hindering Li+ ion transport, switching kinetics, and limiting the films' doping efficiency.

A Multiple-Responsive Fluorescence Probe Based on Water-Soluble Fluorescent Conjugated Polymer for Biomolecules Detection

In this paper, we designed a multiresponsive fluorescent probe based on a synthetic water-soluble conjugated fluorescent polymer (PF-DBT-S-S-PDI) consisting of the conjugated fluorescent backbones and the hydrophilic side chains connected by a disulfide bond. Based on its specific structures, according to two different response mechanisms of fluorescence resonance energy transfer (FRET) and heavy atom effect, the polymer shows high sensitivity and selectivity toward reductive species-thiols and oxidizing substances (such as H2O2) in different ways. Owing to the presence of disulfide bonds, the reductive mercaptan in the solution would cause the water-soluble branches in the micellar structure to break off and leave, which causes the hydrophobic framework of the polymer to aggregate and enhance the fluorescence intensity. On the other hand, because the PDIs in branches are ionic polymers with many iodide ions, H2O2 in solution oxidizes iodide ions into iodide so that the heavy atomic effect of iodide would quench the fluorescence. Both mechanisms have high sensitivity; e.g., the detection limits for mercaptan (such as cysteine) were 0.052 mM, 5.26 μM for H2O2, and 1.06 μM for glucose (combined with oxidase). The multiple detection ability of the polymer has been validated by evaluating free thiols and glucose levels in food samples and tissue homogenates. Our work provided a strategy for integrating multiple species detection capabilities on a single probe.

Spiropyran-containing water-soluble and photoreversible copolymers

Photoreversibility in polymer design enables the control of physical properties using light, and in concert with water-solubility permits applications in aqueous environments. Inspired by environmental application, the primary goal of this paper is to develop a new synthesis pathway and to characterize the structure and functional behavior of spiropyran-containing photoreversible and water-soluble polymers. A one-step facile acylation reaction yielded the spiropyran-containing acrylate (SPA) monomer. Free radical copolymerization afforded a series of copolymers of SPA and oligo(ethylene oxide) monoacrylate monomer (OEOA) (Mn = 480 g/mol). Thermal analysis, including thermogravimetric analysis and differential scanning calorimetry, revealed the influence of SPA content on copolymer weight loss profile and OEOA side chain crystallization/melting. Copolymers exhibited good water solubility owing to the hydrophilic OEOA side chain. Solution rheology unveiled a shear-thickening effect related to the SPA pendent group and revealed the solution viscosity dependence on polymer concentration in aqueous solutions. In water, the designed spiropyran-containing polymers displayed photoreversibility and exhibited high photo-fatigue resistance with >90% of UV–vis absorbance preserved after five cycles. Preliminary Cu2+ complexation studies with SPA-co-OEOA copolymers indicated 88% removal of Cu2+ after UV irradiation, which suggests opportunities as light-responsive polymers for Cu2+ removal.

Low Vanadium Permeability Membranes Based on Flexible Hydrophilic Side Chain Grafted Polybenzimidazole/Polymeric Ionic Liquid for VRFBs

Based on amino polybenzimidazoles with flexible hydrophilic side chains (AmPBI-MOE) and polymeric ionic liquid (PIL), a series of composite membranes (AmPBI-MOE-PIL-X) were fabricated for vanadium redox flow battery applications. Here, 1-Bromo-2-(2-methoxyethoxy)ethane was grafted onto amino polybenzimidazole (AmPBI) by the method of halogenated hydrocarbons, and PIL was synthesized from ionic liquids by in situ radical polymerization to build a hydrogen-bonded cross-linked network within the film. The hydrophilic side chain improves the proton conductivity. With the increase in ionic liquids, the vanadium transmittance and the proton conductivity increase. The AmPBI-MOE-PIL-5 membrane not only exhibits a vanadium ions permeability of 0.88 × 10−9 cm2 min−1, which is much lower than Nafion117 (6.07 × 10−8 cm2 min−1), but also shows a very excellent blocking ability for vanadium ion. The AmPBI-MOE-PIL-5 membrane shows excellent performances at 60 mA cm−2, with VE of 87.93% and EE of 82.87%, both higher than that of Nafion117 membrane in VRFB.

Polyaryl piperidine anion exchange membranes with hydrophilic side chain

Recently, the development of high-performance and durable anion exchange membranes has been a top priority for anion exchange membrane fuel cells. Here, a series of polyaryl piperidine anion exchange membranes with hydrophilic side chain (qBPBA-80-OQ-x) are prepared by the superacid-catalyzed Friedel-Crafts reaction. AFM images show that the hydrophilic side chain and hydrophobic main chain form a distinct microphase separation structure. The AEMs of qBPBA-80-OQ-100 and qBPBA-80 have close mechanical strength, but the ionic conductivity of the former (81 mS/cm, 80 °C) is higher than the latter (73 mS/cm, 80 °C). In addition, qBPBA-80-OQ-100 AEM loses by 15.0% after an alkaline treatment of 720 h, while qBPBA-80 AEM loses by 17.8%. The results indicate that the introduction of hydrophilic side chain not only promotes the formation of microphase separation structure, but also improves the ionic conductivity and alkaline resistance of polyaryl piperidine AEMs.

Hydration of a Side-Chain-Free n-Type Semiconducting Ladder Polymer Driven by Electrochemical Doping

We study the organic electrochemical transistor (OECT) performance of the ladder polymer poly(benzimidazobenzophenanthroline) (BBL) in an attempt to better understand how an apparently hydrophobic side-chain-free polymer is able to operate as an OECT with favorable redox kinetics in an aqueous environment. We examine two BBLs of different molecular masses from different sources. Regardless of molecular mass, both BBLs show significant film swelling during the initial reduction step. By combining electrochemical quartz crystal microbalance gravimetry, in-operando atomic force microscopy, and both ex-situ and in-operando grazing incidence wide-angle X-ray scattering (GIWAXS), we provide a detailed structural picture of the electrochemical charge injection process in BBL in the absence of any hydrophilic side-chains. Compared with ex-situ measurements, in-operando GIWAXS shows both more swelling upon electrochemical doping than has previously been recognized and less contraction upon dedoping. The data show that BBL films undergo an irreversible hydration driven by the initial electrochemical doping cycle with significant water retention and lamellar expansion that persists across subsequent oxidation/reduction cycles. This swelling creates a hydrophilic environment that facilitates the subsequent fast hydrated ion transport in the absence of the hydrophilic side-chains used in many other polymer systems. Due to its rigid ladder backbone and absence of hydrophilic side-chains, the primary BBL water uptake does not significantly degrade the crystalline order, and the original dehydrated, unswelled state can be recovered after drying. The combination of doping induced hydrophilicity and robust crystalline order leads to efficient ionic transport and good stability.

Role of aggregates and microstructure of mixed-ionic-electronic-conductors on charge transport in electrochemical transistors.

Synthetic efforts have delivered a library of organic mixed ionic-electronic conductors (OMIECs) with high performance in electrochemical transistors. The most promising materials are redox-active conjugated polymers with hydrophilic side chains that reach high transconductances in aqueous electrolytes due to volumetric electrochemical charging. Current approaches to improve transconductance and device stability focus mostly on materials chemistry including backbone and side chain design. However, other parameters such as the initial microstructure and microstructural rearrangements during electrochemical charging are equally important and are influenced by backbone and side chain chemistry. In this study, we employ a polymer system to investigate the fundamental electrochemical charging mechanisms of OMIECs. We couple in situ electronic charge transport measurements and spectroelectrochemistry with ex situ X-ray scattering electrochemical charging experiments and find that polymer chains planarize during electrochemical charging. Our work shows that the most effective conductivity modulation is related to electrochemical accessibility of well-ordered, interconnected aggregates that host high mobility electronic charge carriers. Electrochemical stress cycling induces microstructural changes, but we find that these aggregates can largely maintain order, providing insights on the structural stability and reversibility of electrochemical charging in these systems. This work shows the importance of material design for creating OMIECs that undergo structural rearrangements to accommodate ions and electronic charge carriers during which percolating networks are formed for efficient electronic charge transport.

Synthesis and in vitro photodynamic activity of aza-BODIPY-based photosensitizers.

Aza-BODIPY dyes have recently come to attention owing to their excellent chemical and photophysical properties. In particular, their absorption and emission maxima can efficiently be shifted to the red or even to the NIR spectral region. On this basis, aza-BODIPY derivatives are widely investigated as fluorescent probes or phototherapeutic agents. Here we report the synthesis of a set of novel aza-BODIPY derivatives as potential photosensitizers for use in photodynamic therapy. Triazolyl derivatives were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition as the key step. In vitro photodynamic activities of the newly synthesized compounds were evaluated on the A431 human epidermoid carcinoma cell line. Structural differences influenced the light-induced toxicity of the test compounds markedly. Compared to the initial tetraphenyl aza-BODIPY derivative, the compound bearing two hydrophilic triethylene glycol side chains showed substantial, more than 250-fold, photodynamic activity with no dark toxicity. Our newly synthesized aza-BODIPY derivative, acting in the nanomolar range, might serve as a promising candidate for the design of more active and selective photosensitizers.

Oligo(ethylene glycol)-incorporated hole transporting polymers for efficient and stable inverted perovskite solar cells

We develop a series of poly(triarylamine)-based polymers with hydrophilic OEG side chains. The DEG-PTAA-based perovskite solar cell shows the highest efficiency of 22.26% with superior operational stability to the other devices.

Structural modulation of insulin by hydrophobic and hydrophilic molecules.

In the bloodstream, insulin interacts with various kinds of molecules, which can alter its structure and modulate its function. In this work, we have synthesized two molecules having extremely hydrophilic and hydrophobic side chains. The effects of hydrophilic and hydrophobic molecules on the binding with insulin have been investigated through a multi-spectroscopic approach. We found that hydrophilic molecules have a slightly higher binding affinity towards insulin. Insulin can bind with the hydrophilic molecules as it binds glucose. The high insulin binding affinity of a hydrophobic molecule indicates its dual nature. The hydrophobic molecule binds at the hydrophobic pocket of the insulin surface, where hydrophilic molecules interact at the polar surface of the insulin. Such binding with the hydrophobic molecule perturbs strongly the secondary structure of the insulin much more in comparison to hydrophilic molecules. Therefore, the stability of insulin decreases in the presence of hydrophobic molecules.

Emulsifying properties of plant-derived polypeptide and their conjugates: a self-consistent-field calculation study of the impact of hydrolysis.

By considering the hydrolysates of soy protein produced by trypsin as an example, the emulsion stabilizing properties of plant-based protein fragments have been investigated theoretically. We apply Self-Consistent-Field (SCF) calculations to determine the colloidal interactions induced between a pair of droplets stabilized by adsorbed layers of various soy protein fragments. The study is extended to conjugates of such polypeptides, formed by covalent bonding with a suitable hydrophilic sidechain (e.g. a polysaccharide). Our results show that the relatively longer fragments, with a greater number of hydrophobic amino acids, will display a stronger degree of adsorption affinity compared to the smaller hydrolysates, even where the latter may have a higher overall ratio of hydrophobic residues. This suggested that the degree of protein hydrolysis should be carefully controlled and limited to modest values to avoid the generation of a large number of short polypeptides, while still sufficient to improve solubility. While the emulsion stabilizing performance of a protein fragment type is strongly dependent on the conformation it adopts on the interface, we find this to be less critical for the conjugated polypeptides. However, we argue that with increasing degree of hydrolysis, many small fragments will not have the chance to form bonds with polysaccharides. It is demonstrated that the abundance of these unreacted polypeptides in the system severely reduces the efficiency of the conjugated longer protein fragments, preventing their presence on the surface of the droplets through competitive adsorption process.

Preparation and Electrochromic Properties of Benzodithiophene-Isoindigo Conjugated Polymers with Oligoethylene Glycol Side Chains.

Functional polymers featuring good processability in non-halogenated, benzene-free green solvents are highly desired due to health and environmental concerns. Herein, a series of novel D-A type conjugated polymers, PBDT-IIDs, are designed and successfully prepared by "green" functionalization of the polymers with highly hydrophilic, highly polar, highly flexible, and biocompatible oligoethylene glycol (OEG) side chains in order to improve the processability. These series polymers are named PBDT-IID2, PBDT-IID3, and PBDT-IID4, respectively, according to the number of oxygen atoms in the side chain. After confirmation by structural characterization, the basic properties of PBDT-IIDs are also investigated. With the increase in the OEG side chain length, the polymer PBDT-IID4 not only has good solubility in the halogen solvent chlorobenzene, but also exhibits excellent solubility in the green halogen-free solvent methyltetrahydrofuran (Me-THF). As a result, the green solvent Me-THF can also be applied to prepare PBDT-IIDs' electrochromic active layers, except for chlorobenzene and toluene. The electrochromism of PBDT IIDs under both positive and negative voltages has a practical application potential. The several controllable switches between dark green and khaki (0-0.6 V) are expected to show great potential in the field of military camouflage. Furthermore, according to the principle of red, green, and blue (RGB) mixing, light blue-green in the reduced state (-1.6 V) can be used in the preparation of complementary ECDs to provide one of the three primary colors (green).

2D Hexagonal Assemblies of Amphiphilic Double‐Helical Poly(phenylacetylene) Homopolymers with Enhanced Circularly Polarized Luminescence and Chiral Self‐Sorting

AbstractTwo‐dimensional (2D) chiral materials have been attracting immense attentions owing to their unique properties. Herein, we successfully developed a unique assembly strategy of amphiphilic homopolymers to construct stable free‐standing 2D chiral nanosheets in solution. The amphiphilic poly(phenylacetylene) (PPA) homopolymers bearing the hydrophobic and hydrophilic dendritic side chains adopt a DNA‐like double‐helical conformation. The regular hexagonal nanosheets were formed in THF/EtOH through nucleation and epitaxial growth. The sizes of the nanosheets can be modulated from nanometers to submillimeters upon varying the ratio of binary solvents, while the thickness is linearly correlated with the molecular weights. The 2D architecture can significantly enhance the CPL of polymers with a high dissymmetry factor ≈0.1. Driven by a discrimination of helical conformation, the PPAs can self‐sort into homochiral 2D nanosheets, as directly visualized by using fluorescent microscopy.