Excitonic Properties of Organic Dye Aggregates: Contribution of Ukrainian Science.

Unexpected discovery that molecules of organic dyes when they form regular structures can change dramatically their light absorption and fluorescence properties were attracting the minds of researchers for more than eight decades. The progress in investigation of this unique phenomenon described in terms of H- and J-aggregation has led to many practical applications. Here the author expresses his personal view on the dramatic story of switching this research area from empirical knowledge to that standing on strong background of molecular exciton theory. The author was a witness of some of these events and acquainted with several great personalities involved. The major trends of future developments are highlighted.

Light Induced Proton Coupled Charge Transfer Triggers Counterion Directional Translocation.

We demonstrate directed translocation of ClO4 - anions from cationic to neutral binding site along the synthetized BPym-OH dye molecule that exhibits coupled excited-state intramolecular proton-transfer (ESIPT) and charge-transfer (CT) reaction (PCCT). The results of steady-state and time-resolved spectroscopy together with computer simulation and modeling show that in low polar toluene the excited-state redistribution of electronic charge enhanced by ESIPT generates the driving force, which is much stronger than by CT reaction itself and provides more informative gigantic shifts of fluorescence spectra signaling on ultrafast ion motion. The associated with ion translocation red-shifted fluorescence band (at 750 nm, extending to near-IR region) appears at the time ~83 ps as a result of electrochromic modulation of PCCT reaction. It occurs at substantial delay to PCCT that displayed fluorescence band at 640 nm and risetime of <200 fs. Thus, it becomes possible to visualize the manifestations of light-triggered ion translocation and of its driving force by fluorescence techniques and to separate them in time and energy domains.

Double-exponential kinetics of binding and redistribution of the fluorescent dyes in cell membranes witness for the existence of lipid microdomains.

New technique of detecting lateral heterogeneity of the plasma membrane of living cells by means of membrane-binding fluorescent dyes is proposed. The kinetics of dye incorporation into the membrane or its lateral diffusion inside the membrane is measured and decomposed into exponential components by means of the Maximum Entropy Method. Two distinct exponential components are obtained consistently in all cases for several fluorescent dyes, two different cell lines and in different types of experiments including spectroscopy, flow cytometry and fluorescence recovery after photobleaching. These components are attributed to the liquid-ordered and disordered phases in the plasma membrane of studied cells in their dynamic equilibrium.

Breaking the Kasha Rule for More Efficient Photochemistry.

This paper provides a systematic review and analysis of different phenomena that violate a basic principle, Kasha's rule, when applied to photochemical reactions. In contrast to the classical route of ultrafast transition to the lowest energy excited state and photochemical reaction starting therein, in some cases, these reactions proceed directly from high-energy excited states. Nowadays, this phenomenon can be observed for a number of major types of excited-state reactions: harvesting product via intersystem crossing; photoisomerizations; bond-breaking; and electron, proton, and energy transfers. We show that specific conditions for their observation are determined by kinetic factors. They should be among the fastest reactions in studied systems, competing with vibrational relaxation and radiative or nonradiative processes occurring in upper excited states. The anti-Kasha effects, which provide an important element that sheds light on the mechanisms of excited-state transformations, open new possibilities of selective control of these reactions for a variety of practical applications. Efficient utilization of excess electronic energy should enhance performance in the systems of artificial photosynthesis and photovoltaic devices. The modulation of the reporting signal by the energy of excitation of light should lead to new technologies in optical sensing and imaging.

Apoptosis and eryptosis: Striking differences on biomembrane level.

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.

Caspase-3 activation decreases lipid order in the outer plasma membrane leaflet during apoptosis: A fluorescent probe study.

In this research we investigate the connection between the cytoplasmic machinery of apoptosis and the plasma membrane organization by studying the coupling of caspase-3 activation and inhibition with PS exposure and the change of lipid order in plasma membrane sensed by a fluorescent membrane probe NR12S. First, we performed in silico molecular dynamics simulations, which suggest that the mechanism of response of NR12S to lipid order may combine both sensitivity to membrane polarity/hydration and change in the fluorophore orientation. Second, cellular studies revealed that upon triggering apoptosis with IPA-3 and camptothecin the NR12S response is similar to that observed after decrease of lipid order induced by cholesterol depletion, 7-ketocholesterol enrichment or sphingomyelin hydrolysis. NR12S response can be influenced by a caspase-3 inhibitor Z-DEVD-FMK. Flow cytometry data further indicate that the NR12S response correlates with the response of FITC-labeled DEVD-FMK peptide and GFP-labeled Annexin V on the whole time scale (0-24h) of apoptosis induction by camptothecin. We conclude that fine changes in lipid order observed by NR12S are coupled with early steps of cellular events in apoptosis.

Super-Resolution Optical Fluctuation Bio-Imaging with Dual-Color Carbon Nanodots.

Success in super-resolution imaging relies on a proper choice of fluorescent probes. Here, we suggest novel easily produced and biocompatible nanoparticles-carbon nanodots-for super-resolution optical fluctuation bioimaging (SOFI). The particles revealed an intrinsic dual-color fluorescence, which corresponds to two subpopulations of particles of different electric charges. The neutral nanoparticles localize to cellular nuclei suggesting their potential use as an inexpensive, easily produced nucleus-specific label. The single particle study revealed that the carbon nanodots possess a unique hybrid combination of fluorescence properties exhibiting characteristics of both dye molecules and semiconductor nanocrystals. The results suggest that charge trapping and redistribution on the surface of the particles triggers their transitions between emissive and dark states. These findings open up new possibilities for the utilization of carbon nanodots in the various super-resolution microscopy methods based on stochastic optical switching.

Photoluminescence of carbon nanodots: dipole emission centers and electron-phonon coupling.

Inorganic carbon nanomaterials, also called carbon nanodots, exhibit a strong photoluminescence with unusual properties and, thus, have been the focus of intense research. Nonetheless, the origin of their photoluminescence is still unclear and the subject of scientific debates. Here, we present a single particle comprehensive study of carbon nanodot photoluminescence, which combines emission and lifetime spectroscopy, defocused emission dipole imaging, azimuthally polarized excitation dipole scanning, nanocavity-based quantum yield measurements, high resolution transmission electron microscopy, and atomic force microscopy. We find that photoluminescent carbon nanodots behave as electric dipoles, both in absorption and emission, and that their emission originates from the recombination of photogenerated charges on defect centers involving a strong coupling between the electronic transition and collective vibrations of the lattice structure.

Rational design of a solvatochromic fluorescent uracil analogue with a dual-band ratiometric response based on 3-hydroxychromone.

Fluorescent nucleoside analogues with strong and informative responses to their local environment are in urgent need for DNA research. In this work, the design, synthesis and investigation of a new solvatochromic ratiometric fluorophore compiled from 3-hydroxychromones (3HCs) and uracil fragments are reported. 3HC dyes are a class of multi-parametric, environment-sensitive fluorophores providing a ratiometric response due to the presence of two well-resolved bands in their emission spectra. The synthesized conjugate demonstrates not only the preservation but also the improvement of these properties. The absorption and fluorescence spectra are shifted to longer wavelengths together with an increase of brightness. Moreover, the two fluorescence bands are better resolved and provide ratiometric responses across a broader range of solvent polarities. To understand the photophysical properties of this new fluorophore, a series of model compounds were synthesized and comparatively investigated. The obtained data indicate that uracil and 3HC fragments of this derivative are coupled into an electronic conjugated system, which on excitation attains strong charge-transfer character. The developed fluorophore is a prospective label for nucleic acids. Abstract in Ukrainian: .

Fluorescent carbon nanomaterials: "quantum dots" or nanoclusters?

Despite many efforts, the mechanisms of light absorption and emission of small fluorescent carbon nanoparticles (C-dots) are still unresolved and are a subject of active discussion. In this work we address the question as to whether the fluorescence is a collective property of these nanoparticles or they are composed of assembled individual emitters. Selecting three types of C-dots with "violet", "blue" and "green" emissions and performing a detailed study of fluorescence intensity, lifetime and time-resolved anisotropy as a function of excitation and emission wavelengths together with the effect of viscogen and dynamic fluorescence quencher, we demonstrate that the C-dots represent assemblies of surface-exposed fluorophores. They behave as individual emitters, display electronic anisotropy, do not exchange their excited-state energies via homo-FRET and possibly display sub-nanosecond intra-particle mobility.

Excited-state proton coupled charge transfer modulated by molecular structure and media polarization.

Charge and proton transfer reactions in the excited states of organic dyes can be coupled in many different ways. Despite the complementarity of charges, they can occur on different time scales and in different directions of the molecular framework. In certain cases, excited-state equilibrium can be established between the charge-transfer and proton-transfer species. The interplay of these reactions can be modulated and even reversed by variations in dye molecular structures and changes of the surrounding media. With knowledge of the mechanisms of these processes, desired rates and directions can be achieved, and thus the multiple emission spectral features can be harnessed. These features have found versatile applications in a number of cutting-edge technological areas, particularly in fluorescence sensing and imaging.

Michael Kasha: from photochemistry and flowers to spectroscopy and music.

A brilliant scientist and an outstanding personality who was one of the founders of modern photochemistry-Michael Kasha-is the subject of this Essay. Kasha's rule and the Kasha effect both bear his name, and he also discovered the chemical production of singlet molecular oxygen, and was a pioneer of excited-state proton transfer systems. Kasha combined his passion for chemistry and physics with that for music, photography, and botany.

On heterogeneity in fluorescent few-atom silver nanoclusters.

Fluorescent clusters of several Ag atoms are fluorophores with a large set of attractive properties including sub-nanometer size, high fluorescence quantum yield and large Stokes shift. When formed on polymeric scaffolds they exhibit a broad range of heterogeneities in excitation and emission spectra. Addressing this issue we provide detailed spectroscopic studies of these clusters produced by photochemical reduction of silver salt on amphiphilic polystyrene-block-poly(methacrylic acid) block copolymer in N,N-dimethylformamide. Our results allow discriminating three types of emitters differing in positions of their excitation and emission bands. In addition, each band exhibits characteristic features of inhomogeneous broadening, such as strong dependences of excitation spectra on emission wavelength and of emission spectra on excitation wavelength in a mirror-image manner. The latter dependence does not appear on excitation at the 2nd band in the near-UV region. A qualitative model based on inhomogeneous broadening of spectra is suggested to explain these effects.

Proton transfer reactions: From photochemistry to biochemistry and bioenergetics.

The present Review is an attempt by projecting the basic knowledge on photochemical proton transfer to achieve consistent understanding of proton motions in biocatalysis, photobiocatalysis, operation of selective proton channels and systems of photosynthesis and cellular respiration. The basic mechanisms of proton transfer are in active research in the electronic excited states of organic molecules. This allows observing the reactions directly in real time, providing their dynamic and thermodynamic description and coupling with structural and energetic variables. These achievements lay the background for understanding the proton transfers in biochemical reactions, where such ultrafast events are not only 'optically silent' but are hidden under much slower rate-limiting steps, such as protein conformational changes, substrate binding and product release. The mechanistic description of biocatalytic and transmembrane proton transport is shown as a multi-step proton migration that is available for modeling in photochemical reactions. For explaining the formation of transmembrane proton gradients, a simple 'proton lift' concept is presented that may be the basis of further research and analysis.

Dual emission and itsλ-ratiometric detection in analytical fluorimetry Pt. II. Exploration in sensing and imaging.

The wavelength-ratiometric techniques demonstrate strong advantages in fluorescence sensing and imaging over techniques employing variations of intensity at single wavelength. We present different possibilities for realization of these advantages in different simplified, miniaturized and multiplexing devices. They include the smartphone-based detection systems and strips, in which the color changes are observed with naked eye. The array-based techniques and different immunoassays withλ-ratiometric detection demonstrate strongly increased stability and sensitivity. The application areas extend from on-site monitoring of environment and point-of-care diagnostics to testing in personal need. Selected examples of sensing different analytes in chemical and biological systems demonstrate multiple possibilities of coupling the analyte-sensor interaction with the generation ofλ-ratiometric output signal. Among them, simultaneous detection of several analytes and performing logical operations that can be useful in analysis. Finally, the benefits of multicolor ratiometric fluorescence imaging are demonstrated by visualization the functionally important parameters of biological membranes.

Dual emission and itsλ-ratiometric detection in analytical fluorimetry. Pt. I. Basic mechanisms of generating the reporter signal.

The wavelength-ratiometric techniques gain increasing popularity in fluorescence probing and sensing for providing inner reference to output signal and removing instrumental artefacts, in this way increasing the sensitivity and reliability of assays. Recent developments demonstrate that such approach can allow achieving much more, with the application of broad range of novel molecular and nanoscale fluorophores (luminophores), exploring the whole power of photophysical and photochemical effects and using extended range of assay formats. Simplicity of detection and potentially rich content of output data allows realizing these techniques in different simplified, miniaturized and multiplexing devices. The latter issues are discussed in Pt. II of these series.

How cholesterol is distributed between monolayers in asymmetric lipid membranes.

The distribution of cholesterol in asymmetric lipid bilayers was studied by extensive coarse-grained molecular dynamics simulations. The effects of the lipid head group charge, acyl chain saturation, spontaneous membrane curvature and surface tension of the membrane were investigated. Four asymmetric bilayers containing DOPC, DOPS, DSPC or DSPS lipids were simulated on a time scale extended to tens of microseconds. We show that cholesterol strongly prefers anionic lipids to neutral and saturated lipid tails to unsaturated with a distribution ratio of ~0.7 in neutral/anionic bilayers and of ~0.4 in unsaturated/saturated bilayers. Multiple flip-flop transitions of cholesterol were observed directly, and their mean times ranged from 80 to 250 ns. It was shown that the distribution of cholesterol in the asymmetric membrane depends not only on the type of lipid, but also on the local membrane curvature and the surface tension. The membrane curvature enhances the influence of the lipid head groups on cholesterol distribution, while non-optimal surface tension caused by different areas per lipid in different monolayers increases the effect of the lipid tail saturation. It was clearly seen that the monolayers of asymmetric bilayers are interdependent. Mean distances from the bilayer center to cholesterol molecules depend not only on the type of the lipid in the considered monolayer but also on the composition of the opposite monolayer.

The ability of carbon nanoparticles to increase transmembrane current of cations coincides with impaired synaptic neurotransmission.

Here, carbon nanodots synthesized from ß-alanine (Ala-CDs) and detonation nanodiamonds (NDs) were assessed using (1) radiolabeled excitatory neurotransmitters L-[14C]glutamate, D-[2,33H]aspartate, and inhibitory ones [3H]GABA, [3H]glycine for registration of their extracellular concentrations in rat cortex nerve terminals; (2) the fluorescent ratiometric probe NR12S and pH-sensitive probe acridine orange for registration of the membrane lipid order and synaptic vesicle acidification, respectively; (3) suspended bilayer lipid membrane (BLM) to monitor changes in transmembrane current. In nerve terminals, Ala-CDs and NDs increased the extracellular concentrations of neurotransmitters and decreased acidification of synaptic vesicles, whereas have not changed sufficiently the lipid order of membrane. Both nanoparticles, Ala-CDs and NDs, were capable of increasing the conductance of the BLM by inducing stable potential-dependent cation-selective pores. Introduction of divalent cations, Zn2+ or Cd2+ on the particles` application side (cis-side) increased the rate of Ala-CDs pore-formation in the BLM. The application of positive potential (+100 mV) to the cis-chamber with Ala-CDs or NDs also activated the insertion as compared with the negative potential (-100 mV). The Ala-CD pores exhibited a wide-range distribution of conductances between 10 and 60 pS and consecutive increase in conductance of each major peak by ~10 pS, which suggest the clustering of the same basic ion-conductive structure. NDs also formed ion-conductive pores ranging from 6 pS to 60 pS with the major peak of conductance at ~12 pS in cholesterol-containing membrane. Observed Ala-CDs and NDs-induced increase in transmembrane current coincides with disturbance of excitatory and inhibitory neurotransmitter transport in nerve terminals.

Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: effects of cholesterol depletion and apoptosis.

Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.

The concept of λ-ratiometry in fluorescence sensing and imaging.

Very limited number of parameters is available for fluorescence sensing and imaging. The changes of intensity are of low analytical value due to the absence of internal reference. Anisotropy and lifetime sensing have their own limitations. In this respect the λ-ratiometric (based on intensity ratios at two or more wavelengths) recording of spectral changes becomes more popular. Because the spectral changes are connected directly with the variations of interaction energies this approach is seen as the most universal method to study intermolecular interactions. It is applicable for different sensor formats and for obtaining analytical information from cell images. Here we critically analyze different approaches in λ-ratiometric sensing that use single and double fluorescence emitters and are based on different mechanisms producing spectroscopic change. Very promising is the exploration of mechanisms that allow obtaining ratiometric response from a single dye.