Jose M. Paredes

Visible Absorption and Fluorescence Spectroscopy of Conformationally Constrained, Annulated BODIPY Dyes

Six conformationally restricted BODIPY dyes with fused carbocycles were synthesized to study the effect of conformational mobility on their visible electronic absorption and fluorescence properties. The symmetrically disubstituted compounds (2, 6) have bathochromically shifted absorption and fluorescence spectral maxima compared to those of the respective asymmetrically monosubstituted dyes (1, 5). Fusion of conjugation extending rings to the α,β-positions of the BODIPY core is an especially effective method for the construction of boron dipyrromethene dyes absorbing and emitting at longer wavelengths. The fluorescence quantum yields Φ of dyes 1-6 are high (0.7 ≤ Φ ≤ 1.0). The experimental results are backed up by quantum chemical calculations of the lowest electronic excitations in 1, 2, 5, 6, and corresponding dyes of related chemical structure but without conformational restriction. The effect of the molecular structure on the visible absorption and fluorescence emission properties of 1-6 has been examined as a function of solvent by means of the recent, generalized treatment of the solvent effect, proposed by Catalán (J. Phys. Chem. B 2009, 113, 5951-5960). Solvent polarizability is the primary factor responsible for the small solvent-dependent shifts of the visible absorption and fluorescence emission bands of these dyes.

Real-time phosphate sensing in living cells using fluorescence lifetime imaging microscopy (FLIM).

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dyes nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

Tuned lifetime, at the ensemble and single molecule level, of a xanthenic fluorescent dye by means of a buffer-mediated excited-state proton exchange reaction

The photophysical behaviour of the new fluorescein derivative 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one has been explored by using absorption, and steady-state, time-resolved and single-molecule fluorescence measurements. The apparent ground-state acidity constant of the dye determined by both the absorbance and steady-state fluorescence is almost independent of the added buffer and salt concentrations. The excited-state proton exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis (GCA) to elucidate the values of the excited-state rate constants. The features of this system make the fluorescence decays monoexponential at pH values and phosphate buffer concentrations higher than 6.10 and 0.2 M respectively, with the possibility of tuning the fluorescence lifetime value by changing pH or buffer concentrations. The tuned lifetimes obtained by means of phosphate concentration at constant pH have also been recovered at the single-molecule level.

Dynamics of water-in-oil nanoemulsions revealed by fluorescence lifetime correlation spectroscopy.

The size, diffusional properties, and dynamics of reverse water-in-oil nanoemulsions, or reverse micelles (RMs), have been widely investigated because of interest in this system as a model for biological compartmentalization. Here, we have employed fluorescence lifetime correlation spectroscopy (FLCS) to reveal the dynamics and sizes of aerosol-OT (AOT)/isooctane RMs using a fluorescent xanthene derivative called Tokyo Green II (TG-II). The dye undergoes a partition and a shift in its tautomeric equilibrium such that the TG-II anion remains in the inner micellar aqueous core, and the neutral quinoid form lies in the interfacial region. By applying FLCS, we specifically obtained the lifetime filtered autocorrelation curves of the anionic TG-II, which shows a characteristic lifetime of approximately 4 ns. Analysis of the FLCS curves provides the diffusion coefficient and hydrodynamic radius of the RMs as well as micelle dynamics in the same experiment. The FLCS curves show dynamics in the microsecond time range, which represents an interconversion rate that changes the distribution of the TG-II neutral and anionic forms in the hydrophobic interface and the water core.

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.

New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Photophysics of the interaction between a fluorescein derivative and Ficoll.

Ficoll has been widely used as a crowding agent to mimic intracellular media because it is believed to be noninteracting and is composed of mixed sizes such that smaller and larger diffusing solutes can be studied. Due to the interest that the fluorescent dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (TG-II) as a fluorometric probe of phosphate ions in intracellular media could generate, we describe the spectral characteristics of the system TG-II-Ficoll in aqueous solution by means of absorption spectroscopy, steady-state fluorescence, time-resolved fluorescence, time-resolved emission spectroscopy, and fluorescence lifetime correlation spectroscopy. The spectral characteristics found are consistent with the formation of an adsorption complex on the surface of Ficoll, probably due to hydrogen bonding between TG-II and Ficoll. In addition, the diffusion coefficient calculated for the association was similar to the diffusion coefficient previously recovered for Ficoll in the same experimental conditions. Therefore, our overall data clearly demonstrate that Ficoll is not an inert crowding agent when in the presence of fluorescein derivative dyes.

Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye.

Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, was determined by absorbance and steady-state fluorescence at very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton-transfer (ESPT) reaction among the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, the values of the excited-state rate constants were determined by global analysis of the fluorescence decay traces recorded as a function of pH, and buffer concentration. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.

Two-Step Amyloid Aggregation: Sequential Lag Phase Intermediates

The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation.

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants.