A Case-Study on the Photophysics of Chalcogen-Substituted Zinc(II) Phthalocyanines.

Singlet dioxygen has been widely applied in different disciplines such as medicine (photodynamic therapy or blood sterilization), remediation (wastewater treatment) or industrial processes (fine chemicals synthesis). Particularly, it can be conveniently generated by energy transfer between a photosensitizer's triplet state and triplet dioxygen upon irradiation with visible light. Among the best photosensitizers, substituted zinc(II) phthalocyanines are prominent due to their excellent photophysical properties, which can be tuned by structural modifications, such as halogen- and chalcogen-atom substitution. These patterns allow for the enhancement of spin-orbit coupling, commonly attributed to the heavy atom effect, which correlates with the atomic number ( Z ${Z}$ ) and the spin-orbit coupling constant ( ζ ${\zeta }$ ) of the introduced heteroatom. Herein, a fully systematic analysis of the effect exerted by chalcogen atoms on the photophysical characteristics (absorption and fluorescence properties, lifetimes and singlet dioxygen photogeneration), involving 30 custom-made ß-tetrasubstituted chalcogen-bearing zinc(II) phthalocyanines is described and evaluated regarding the heavy atom effect. Besides, the intersystem crossing rate constants are estimated by several independent methods and a quantitative profile of the heavy atom is provided by using linear correlations between relative intersystem crossing rates and relative atomic numbers. Good linear trends for both intersystem crossing rates (S1-T1 and T1-S0) were obtained, with a dependency on the atomic number and the spin-orbit coupling constant scaling as Z 0 . 4 ${{Z}^{0.4}}$ and ζ 0 . 2 ${{\zeta }^{0.2}}$ , respectively The trend shows to be independent of the solvent and temperature.

Operando detection and suppression of spurious singlet oxygen in Li-O2 batteries.

The rechargeable lithium air (oxygen) battery (Li-O2) has very high energy density, comparable to that of fossil fuels (∼3600 W h kg-1). However, the parasitic reactions of the O2 reduction products with solvent and electrolyte lead to capacity fading and poor cyclability. During the oxygen reduction reaction (ORR) in aprotic solvents, the superoxide radical anion (O2˙-) is the main one-electron reaction product, which in the presence of Li+ ions undergoes disproportionation to yield Li2O2 and O2, a fraction of which results in singlet oxygen (1O2). The very reactive 1O2 is responsible for the spurious reactions that lead to high charging overpotential and short cycle life due to solvent and electrolyte degradation. Several techniques have been used for the detection and suppression of 1O2 inside a Li-O2 battery under operation and to test the efficiency and electrochemical stability of different physical quenchers of 1O2: azide anions, 1,4-diazabicyclo[2.2.2]octane (DABCO) and triphenylamine (TPA) in different solvents (dimethyl sulfoxide (DMSO), diglyme and tetraglyme). Operando detection of 1O2 inside the battery was accomplished by following dimethylanthracene fluorescence quenching using a bifurcated optical fiber in front-face mode through a quartz window in the battery. Differential oxygen-pressure measurements during charge-discharge cycles vs. charge during battery operation showed that the number of electrons per oxygen molecule was n > 2 in the absence of physical quenchers of 1O2, due to spurious reactions, and n = 2 in the presence of physical quenchers of 1O2, proving the suppression of spurious reactions. Battery cycling at a limited specific capacity of 500 mA h gC-1 for the MWCNT cathode and 250 mA gC-1 current density, in the absence and presence of a physical quencher or a physical quencher plus the redox mediator I3-/I- (with a lithiated Nafion® membrane), showed increasing cyclability according to coulombic efficiency and cell voltage data over 100 cycles. Operando Raman studies with a quartz window at the bottom of the battery allowed detection of Li2O2 and excess I3- redox mediator during discharge and charge, respectively.

Photoactive Red Fluorescent SiO2 Nanoparticles Based on Controlled Methylene Blue Aggregation in Reverse Microemulsions.

We present a reverse microemulsion synthesis procedure for incorporating methylene blue (MB), a known FDA-approved type-II red-absorbing photosensitizer and 1O2 generator, into the matrix of hydrophobic-core/hydrophilic-shell SiO2 nanoparticles. Different synthesis conditions were explored with the aim of controlling the entrapped-dye aggregation at high dye loadings in the hydrophobic protective core; minimizing dye aggregation ensured highly efficient photoactive nanoentities for 1O2 production. Monitoring the synthesis in real time using UV-vis absorption allowed tracking of the dye aggregation process. In particular, silica nanoparticles (MB@SiO2 NPs) of ∼50 nm diameter size with a high local entrapped-MB concentration (∼10-2 M, 1000 MB molecules per NP) and a moderate proportion of dye aggregation were obtained. The as-prepared MB@SiO2 NPs showed a high singlet oxygen photogeneration efficiency (ΦΔ = 0.30 ± 0.05), and they can be also considered as red fluorescent probes (ΦF ∼ 0.02, λmax ∼ 650 nm). The distinctive photophysical and photochemical characteristics of the synthesized NPs reveal that the reverse microemulsion synthesis procedure offers an interesting strategy for the development of complex theranostic nano-objects for photodynamic therapy.

Photophysics at Unusually High Dye Concentrations.

The study of the interaction of light with systems at high dye concentrations implies a great challenge because several factors, such as emission reabsorption, dye aggregation, and energy trapping, hinder rationalization and interpretation of the involved photophysical processes. Space constraints induce dye interaction even in the absence of ground state stabilization of dimers and oligomers. At distances on the order of 1 nm, statistical energy traps are usually observed. At longer distances, excited state energy transfer takes place. Most of these factors do not result in ground state spectroscopic changes. Rather, fluorescence phenomena such as inner filter effects, concentration-dependent Stokes' shifts, and changes in quantum yields and decay times are evidenced. Photophysical studies are commonly carried out at high dilution, to minimize dye-dye interactions and emission reabsorption, and in the absence of light scattering. Under these conditions, the physical description of the system becomes rather simple. Fluorescence and triplet quantum yields become molecular properties and can be easily related to ratios of rate constants. However, many systems containing dyes able to fulfill specific functions, whether man-made or biological, are far from being dilute and scattering-free. The photosynthetic apparatus is a paradigmatic example. It is clear that isolation of components allows gathering relevant information about complex systems. However, knowledge of the photophysical behavior in the unaltered environment is essential in most cases. Complexity generally increases when light scattering is present. Despite that, our experience shows that light scattering, when correctly handled, may even simplify the task of unraveling molecular parameters. We show that methods and models aiming at the determination and interpretation of fluorescence and triplet quantum yields as well as energy transfer efficiencies can be developed on the basis of simple light-scattering theories. Photophysical studies were extended to thin films and layer-by-layer assemblies. Procedures are presented for the evaluation of fluorescence reabsorption in concentrated fluid solutions up to the molar level, which are being applied to ionic liquids, in which the emitting species are the bulk ions. Fluorescence reabsorption models proved to be useful in the interpretation of the photophysics of living organisms such as plant leaves and fruits. These new tools allowed the assessment of chlorophyll fluorescence at the chloroplast, leaf and canopy levels, with implications in remote sensing and the development of nondestructive optical methods.

Photophysics of Xanthene Dyes at High Concentrations in Solid Environments: Charge Transfer Assisted Triplet Formation.

The photophysical behavior of two xanthene dyes, Eosin Y and Phloxine B, included in microcrystalline cellulose particles is studied in a wide concentration range, with emphasis on the effect of dye concentration on fluorescence and triplet quantum yields. Absolute fluorescence quantum yields in the solid-state were determined by means of diffuse reflectance and steady-state fluorescence measurements, whereas absolute triplet quantum yields were obtained by laser-induced optoacoustic spectroscopy and their dependence on dye concentration was confirmed by diffuse reflectance laser flash photolysis and time-resolved phosphorescence measurements. When both quantum yields are corrected for reabsorption and reemission of radiation, ΦF values decrease strongly on increasing dye concentration, while a less pronounced decay is observed for ΦT . Fluorescence concentration quenching is attributed to the formation of dye aggregates or virtual traps resulting from molecular crowding. Dimeric traps are however able to generate triplet states. A mechanism based on the intermediacy of charge-transfer states is proposed and discussed. Calculation of parameters for photoinduced electron transfer between dye molecules within the traps evidences the feasibility of the proposed mechanism. Results demonstrate that photoactive energy traps, capable of yielding dye triplet states, can be formed even in highly-concentrated systems with random dye distributions.

Rose bengal in poly(2-hydroxyethyl methacrylate) thin films: self-quenching by photoactive energy traps.

The effect of dye concentration on the fluorescence,ΦF, and singlet molecular oxygen,ΦΔ, quantum yields of rose bengal loaded poly(2-hydroxyethyl methacrylate) thin films (∼200 nm thick) was investigated, with the aim of understanding the effect of molecular interactions on the photophysical properties of dyes in crowded constrained environments. Films were characterized by absorption and fluorescence spectroscopy, singlet molecular oxygen (1O2) production was quantified using a chemical monitor, and the triplet decay was determined by laser flash-photolysis. For the monomeric dilute dye, ΦF = 0.05 ± 0.01 and ΦΔ = 0.76 ± 0.14. The effect of humidity and the photostability of the dye were also investigated. Spectral changes in absorption and fluorescence in excess of 0.05 M and concentration self-quenching after 0.01 M are interpreted in the context of a quenching radius model. Calculations of energy migration and trapping rates were performed assuming random distribution of the dye. Best fits of fluorescence quantum yields with concentration are obtained in the whole concentration range with a quenching radius r Q = 1.5 nm, in the order of molecular dimensions. Agreement is obtained only if dimeric traps are considered photoactive, with an observed fluorescence quantum yield ratio ΦF,trap/ΦF,monomer ≈ 0.35. Fluorescent traps are capable of yielding triplet states and 1O2. Results show that the excited state generation efficiency, calculated as the product between the absorption factor and the fluorescence quantum yield, is maximized at around 0.15 M, a very high concentration for random dye distributions. Relevant information for the design of photoactive dyed coatings is provided.

Steady-State Fluorescence of Highly Absorbing Samples in Transmission Geometry: A Simplified Quantitative Approach Considering Reabsorption Events.

A simplified methodology to acquire steady-state emission spectra and quantum yields of highly absorbing samples is presented. The experimental setup consists of a commercial spectrofluorometer adapted to transmission geometry, allowing the detection of the emitted light at 180° with respect to the excitation beam. The procedure includes two different mathematical approaches to describe and reproduce the distortions caused by reabsorption on emission spectra and quantum yields. Toluene solutions of 9,10-diphenylanthracence, DPA, with concentrations ranging between 1.12 × 10-5 and 1.30 × 10-2 M, were used to validate the proposed methodology. This dye has significant probability of reabsorption and re-emission in concentrated solutions without showing self-quenching or aggregation phenomena. The results indicate that the reabsorption corrections, applied on molecular emission spectra and quantum yields of the samples, accurately reproduce experimental data. A further discussion is performed concerning why the re-emitted radiation is not detected in the experiments, even at the highest DPA concentrations.

Tuning the concentration of dye loaded polymer films for maximum photosensitization efficiency: phloxine B in poly(2-hydroxyethyl methacrylate).

Fluorescence and singlet molecular oxygen ((1)O2) quantum yields for phloxine B loaded poly(2-hydroxyethyl methacrylate) thin films are determined at dye concentrations from 0.015 to 22 wt%. Fluorescence self-quenching and the fall off of the (1)O2 quantum yield observed above 0.1 wt% are attributed to very weakly interacting close-lying dye molecules acting as energy traps arising from molecular confinement. The maximum singlet oxygen generation efficiency (quantum yield × absorption factor) lies at concentrations around 2 wt%, where fluorescence self quenching amounts to more than 80%. Data are fitted quantitatively by using a quenching radius model involving energy migration and trapping with rQ = 1.2 nm. The present results constitute a proof of concept for the rational design of heterogeneous photosensitizers in general and, particularly, for applications in which the antimicrobial activity of singlet oxygen is central.

Effect of concentration on the formation of rose bengal triplet state on microcrystalline cellulose: a combined laser-induced optoacoustic spectroscopy, diffuse reflectance flash photolysis, and luminescence study.

Laser-induced optoacoustic spectroscopy (LIOAS), diffuse reflectance laser flash photolysis (DRLFP), and laser-induced luminescence (LIL) have been applied in conjunction to the determination of triplet state quantum yields of Rose Bengal (RB) supported on microcrystalline cellulose, a strongly light-scattering solid. Among the three used methods, the only one capable of providing absolute triplet quantum yields is LIOAS, but DRLFP and LIL aid in demonstrating that the LIOAS signal arises in fact from the triplet state and confirm the trend found with RB concentration. The coherence found for the three techniques demonstrates the usefulness of the approach. Observed triplet quantum yields are nearly constant within a limited concentration range, after which they decay strongly due to the generation of inactive dye aggregates or energy trapping centers. When quantum yields are divided by the fraction of absorbed light exciting the dye, the quotient falls off steadily with concentration, following the same trend as the observed fluorescence quantum yield. The conditions that maximize triplet formation are determined as a compromise between the rising light absorption and the decrease of quantum yield with RB concentration.

Evidence on dye clustering in the sensitization of TiO2 by aluminum phthalocyanine.

As previous studies have shown, the photocatalytic reduction of Cr(VI) to Cr(III) in the presence of 4-chlorophenol can be carried out efficiently under visible irradiation using TiO2 modified with hydroxoaluminum-tricarboxymonoamide phthalocyanine (AlTCPc) in spite of the high aggregation tendency of the dye. In the present work, photocurrent and absorption spectra of AlTCPc modified TiO2 films are studied together with absorption and fluorescence of the dye in solution as a function of the concentration of the dye to clarify (a) the role of aggregates and the nature of the species responsible for electron injection into the semiconductor and (b) the reasons why, as reported earlier, the photocatalytic activity is nearly independent of dye loading at constant TiO2 mass. Results are consistent with the presence of AlTCPc clusters with similar properties both on the TiO2 surface and in H2O-DMSO solution. The actual photoactive species is the monomeric dye electronically coupled to the semiconductor. Monomer concentration depends only slightly on AlTCPc analytical concentration, in a way similar to surfactant monomers in micellar equilibrium, thus explaining the independence of photocatalytic activity on dye concentration.

Effect of concentration on the photophysics of dyes in light-scattering materials.

Photoactive materials based on dye molecules incorporated into thin films or bulk solids are useful for applications as photosensitization, photocatalysis, solar cell sensitization and fluorescent labeling, among others. In most cases, high concentrations of dyes are desirable to maximize light absorption. Under these circumstances, the proximity of dye molecules leads to the formation of aggregates and statistical traps, which dissipate the excitation energy and lower the population of excited states. The search for enhancement of light collection, avoiding energy wasting requires accounting the photophysical parameters quantitatively, including the determination of quantum yields, complicated by the presence of light scattering when particulate materials are considered. In this work we summarize recent advances on the photophysics of dyes in light-scattering materials, with particular focus on the effect of dye concentration. We show how experimental reflectance, fluorescence and laser-induced optoacoustic spectroscopy data can be used together with theoretical models for the quantitative evaluation of inner filter effects, fluorescence and triplet formation quantum yields and energy transfer efficiencies.

Eosin Y triplet state as a probe of spatial heterogeneity in microcrystalline cellulose.

The photophysical behavior of eosin Y adsorbed onto microcrystalline cellulose was evaluated by reflectance spectroscopy, steady-state fluorescence spectroscopy and laser induced time-resolved luminescence. On increasing the concentration of the dye, small changes in absorption spectra, fluorescence redshifts and fluorescence quenching are observed. Changes in absorption spectra point to the occurrence of weak exciton interactions among close-lying dye molecules, whereas fluorescence is affected by reabsorption and excitation energy trapping. Phosphorescence decays are concentration independent as a result of the negligible exciton interaction of dye pairs in the triplet state. Lifetime distribution and bilinear regression analyses of time-resolved phosphorescence and delayed fluorescence spectra reveal the existence of two different environments: long-lived, more energetic triplet states arise from dyes tightly entrapped within the cellulose chains, while short-lived, less-energetic states result from dyes in more flexible environments. Stronger hydrogen bond interactions between the dye and cellulose hydroxyl groups lead in the latter case to a lower triplet energy and faster radiationless decay. These effects, observed also at low temperatures, are similar to those encountered in several amorphous systems, but rather than being originated in changes in the environment during the triplet lifetime, they are ascribed in this case to spatial heterogeneity.

Phloxine B as a probe for entrapment in microcrystalline cellulose.

The photophysical behaviour of phloxine B adsorbed onto microcrystalline cellulose was evaluated by reflectance spectroscopy and laser induced time-resolved luminescence in the picosecond-nanosecond and microsecond-millisecond ranges. Analysis of the absorption spectral changes with concentration points to a small tendency of the dye to aggregate in the range of concentrations under study. Prompt fluorescence, phosphorescence and delayed fluorescence spectral decays were measured at room temperature and 77 K, without the need of sample degassing because cellulose protects triplet states from oxygen quenching. In all cases, spectral changes with time and lifetime distribution analysis were consistent with the dye coexisting in two different environments: dyes tightly entrapped between polymer chains in crystalline regions of cellulose showed longer fluorescence and phosphorescence lifetimes and more energetic triplet states, while dyes adsorbed in more amorphous regions of the support showed shorter lifetimes and less energetic triplet states. This behaviour is discussed in terms of the different dye-support interactions in both kinds of adsorption sites.

Silicon nanoparticle photophysics and singlet oxygen generation.

The effect of molecular oxygen and water on the blue photoluminescence of silicon nanoparticles synthesized by anodic oxidation of silicon wafers and surface functionalized with 2-methyl 2-propenoic acid methyl ester is investigated. The particles of 3 +/- 1 nm diameter and a surface composition of Si(3)O(6)(C(5)O(2)H(8)) exhibit room-temperature luminescence in the wavelength range 300-600 nm upon excitation with 300-400 nm light. The luminescence shows vibronic resolution and high quantum yields in toluene suspensions, while a vibronically unresolved spectrum and lower emission quantum yields are observed in aqueous suspensions. The luminescence intensity, though not the spectrum features, depends on the presence of dissolved O(2). Strikingly, the luminescence decay time on the order of 1 ns does not depend on the solvent or on the presence of O(2). To determine the mechanisms involved in these processes, time-resolved and steady-state experiments are performed. These include low-temperature luminescence, heavy atom effect, singlet molecular oxygen ((1)O(2)) phosphorescence detection, reaction of specific probes with (1)O(2), and determination of O(2) and N(2) adsorption isotherms at 77 K. The results obtained indicate that physisorbed O(2) is capable of quenching nondiffusively the particle luminescence at room temperature. The most probable mechanism for (1)O(2) generation involves the energy transfer from an exciton singlet state to O(2) to yield an exciton triplet of low energy (<0.98 eV) and (1)O(2). In aqueous solutions, excited silicon nanoparticles are able to reduce methylviologen on its surface.

Trapping of Rhodamine 6G excitation energy on cellulose microparticles.

Rhodamine 6G (R6G) was adsorbed on cellulose microparticles and fluorescence quantum yields and decays were measured as a function of dye loading. Though no spectroscopic evidence of dye aggregation was found, a noticeable decrease of quantum yield--after correction for reabsorption and reemission of fluorescence--and shortening of decays were observed at the highest loadings. These effects were attributed to the dissipation of the excitation energy by traps constituted by R6G pairs, leading to static and dynamic quenching produced by direct absorption of traps and non-radiative energy transfer from monomers, respectively. Regarding the nature of traps, two extreme approaches were considered: (a) equilibrium between monomers slightly interacting in the ground state and (b) randomly distributed monomers located below a critical distance (statistical traps). Both approaches accounted quantitatively for the observed facts. The effect of energy migration was evaluated through computational simulations. As the concentration of traps could only be indirectly inferred, in some experiments an external energy transfer quencher, Methylene Blue, was coadsorbed and the results were compared with those obtained with pure R6G.

Photoreduction of Cr(VI) using hydroxoaluminiumtricarboxymonoamide phthalocyanine adsorbed on TiO2.

Hydroxoaluminiumtricarboxymonoamide phthalocyanine (AlTCPc) adsorbed at different loadings on TiO(2) Degussa P-25 was tested for Cr(vi) photocatalytic reduction under visible irradiation in the presence of 4-chlorophenol (4-CP) as sacrificial donor. A rapid reaction takes place in spite of the presumable aggregation of the dye on the TiO(2) surface. The removal of Cr(vi) is fairly negligible under visible-light irradiation, either without photocatalyst or in the presence of bare TiO(2). The fast capture of conduction band electrons by Cr(vi), which forms a surface complex with TiO(2), inhibits the formation of reactive oxygen species in the reductive pathway. This fact and the easier oxidation of 4-CP as compared to AlTCPc hinder the photobleaching of the dye and make feasible Cr(vi) reduction under visible irradiation. The consumption of Cr(vi) follows a pseudo-first order kinetics; the decay constant depends, in the studied range, on the photocatalyst mass, but it is barely affected by dye loading. The presence of 4-CP is essential, but its concentration has no effect on the Cr(vi) decay rate. Oxidation products of 4-CP, such as hydroquinone, catechol or benzoquinone, are not observed. Direct evidence of the one-electron reduction of Cr(vi) to Cr(v) was obtained by EPR spectroscopy using citric acid as Cr(v) trapping agent. In this case, disappearance of Cr(v) also follows a first order decay, but conduction band electrons do not seem to be involved. The fact that oxidation products of 4-CP are not observed is consistent with the fast dark removal of reaction intermediates by Cr(v), proved by EPR.

Pitfalls and limitations in the practical use of Förster's theory of resonance energy transfer.

Disparate presentations in the literature of the basic equations of Förster's theory of resonance energy transfer are clarified and the limitations of these equations are discussed.

Excitation energy transfer and trapping in dye-loaded solid particles.

The photophysics of several systems composed of a single dye or pairs of dyes attached to solid particles has been studied in the dry solid state at high dye concentrations taking into account light scattering and inner filter effects. Interaction among dye molecules and singlet-singlet energy transfer are relevant in these conditions, as has been demonstrated for pairs of dyes with suitable spectral overlap. For single dyes, after correction for radiative energy transfer, fluorescence quenching is observed as the surface concentration increases. This effect is explained by two different trapping models. Irrespective of the nature of the traps, concentration quenching may be of static (trap absorption) and dynamic (energy transfer) nature. The unraveling of energy trapping mechanisms is a key to the development of efficient photoactive solid materials.

Energy transfer from chemically attached rhodamine 101 to adsorbed methylene blue on microcrystalline cellulose particles.

Rhodamine 101 (R101) was chemically attached onto microcrystalline cellulose and methylene blue (MB) was adsorbed to a sample bearing nearby 6 x 10(-7) mol R101 (g cellulose)(-1). The system was studied by reflectance and emission spectroscopy in the solid state. R101 shows no aggregation in these conditions and, while pure MB builds up dimers on cellulose even at 2 x 10(-8) mol g(-1), in the presence of R101 no evidence on selfaggregation or heteroaggregation is found up to around 10(-6) mol g(-1). No exciplex formation is found as well. The overall fluorescence quantum yield measured on thick layers, once re-absorption effects are accounted for, amounts to 0.80 +/- 0.07 for pure R101 and decreases steadily on increasing the concentration of MB. Results demonstrate the occurrence of radiative and nonradiative singlet energy transfer from R101 to MB. For thick layers of particles, the combined effect of both kinds of energy transfer amounts to nearly 80% at the highest acceptor concentration, while nonradiative transfer reaches 60% both for thin and optically thick layers. The dependence of nonradiative energy transfer efficiencies on the acceptor concentration is analyzed and the origin of departures from Förster behavior at low acceptor concentration is discussed.

Energy transfer among dyes on particulate solids.

Absorption and fluorescence properties of methylene blue (MB), a well-known singlet molecular oxygen photosensitizer, and its mixtures with pheophorbide-a (Pheo) sorbed on microgranular cellulose are studied, with emphasis on radiative and nonradiative energy transfer from Pheo to MB. Although pure MB builds up dimeric species on cellulose even at 2 x 10(-8) mol g(-1), addition of 2.05 x 10(-7) mol g(-1) Pheo largely inhibits aggregation up to nearly 10(-6) mol g(-1) MB. At the same time, the absorption spectrum of monomeric MB in the presence of Pheo differs from the spectrum in pure cellulose. Both effects reveal a strong influence of Pheo on the medium properties. A model relying entirely on experimental data is developed, through which energy transfer efficiencies can be calculated for thin and thick layers of dye-loaded cellulose. At the largest concentration of MB assuring no dye aggregation, nonradiative energy transfer efficiencies reach a maximum value of nearly 40%. This value is quite high, taking into account the low fluorescence quantum yield of Pheo, Phi = 0.21, and results from the existence of high local concentrations of the acceptor within the supporting material. These results show that large energy transfer rates can exist in a system devoid of any special molecular organization.