Fluorescence Lifetime Imaging Microscopy for the Detection of Intracellular pH with Quantum Dot Nanosensors

While the use of quantum dot (QD) nanoparticles for bioimaging and sensing has been improved and exploited during the last several years, most studies have used emission intensity-based techniques. Fluorescence lifetime imaging microscopy (FLIM) can also be employed for sensing purposes, overcoming many of the limitations of the aforementioned systems. Herein, we show that the photoluminescence (PL) lifetime of mercaptopropionic acid-capped QDs (MPA-QDs) collected from FLIM images can be used to determine intracellular pH. The PL average lifetime of MPA-QDs varied from 8.7 ns (pH < 5) to 15.4 ns (pH > 8) in media mimicking the intracellular environment. These long decay times of QD nanoparticles make them easily distinguishable from intrinsic cell autofluorescence, improving selectivity in sensing applications. We demonstrate, for the first time, the successful detection of changes in the intracellular pH of different cell types by examining the PL decay time of QDs. In particular, the combination of FLIM methodologies with QD nanoparticles exhibits greatly improved sensitivity compared with other fluorescent dyes for pH imaging. A detailed description of the advantages of the FLIM technique is presented

The First Step of Amyloidogenic Aggregation

The structural and dynamic characterization of the on-pathway intermediates involved in the mechanism of amyloid fibril formation is one of the major remaining biomedical challenges of our time. In addition to mature fibrils, various oligomeric structures are implicated in both the rate-limiting step of the nucleation process and the neuronal toxicity of amyloid deposition. Single-molecule fluorescence spectroscopy (SMFS) is an excellent tool for extracting most of the relevant information on these molecular systems, especially advanced multiparameter approaches, such as pulsed interleaved excitation (PIE). In our investigations of an amyloidogenic SH3 domain of α-spectrin, we have found dynamic oligomerization, even prior to incubation. Our single-molecule PIE experiments revealed that these species are small, mostly dimeric, and exhibit a loose and dynamic molecular organization. Furthermore, these experiments have allowed us to obtain quantitative information regarding the oligomer stability. These pre-amyloidogenic oligomers may potentially serve as the first target for fibrillization-prevention strategies

Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO‑3 Intercalator Dye

We report a thorough photophysical characterization of the interactions between double-stranded DNA (dsDNA) and the trimethine cyanine homodimer dye YOYO-3. The fluorescence emission of this dye is enhanced by intercalation within the DNA double helix. We have explored the saturation of the dsDNA by bound YOYO-3 at the single-molecule level by studying the single-pair Förster resonance energy transfer (FRET) from an energy donor, Alexa Fluor 488, tagged at the 5′ end of the double helix and the energy acceptor, YOYO-3, bound to the same DNA molecule. The spontaneous binding of YOYO-3 gives rise to an effective distribution of different FRET efficiencies and, therefore, donor–acceptor (D–A) distances. These distributions reveal the existence of multiple states of YOYO-3. Steady-state and time-resolved fluorescence and circular dichroism confirmed the presence of a DNA-bound aggregate of YOYO-3, conspicuous at high dye/base pair ratios. The spectral features of the aggregate suggest that it may have the structure of a parallel H-aggregate

Interaction of YOYO‑3 with Different DNA Templates to Form H‑Aggregates

Homodimeric cyanine dyes are DNA intercalators that display a large enhancement of fluorescence emission when bound to double-stranded DNA. However, other different interaction modes are possible, such as H-type molecular aggregates of the dye, templated by the nucleic acid. In this paper, we study in depth the formation of nonfluorescent H-aggregates of the cyanine homodimer YOYO-3 with two different DNA templates using absorption and both steady-state and time-resolved fluorescence spectroscopy. First, a nonfluorescent YOYO-3 H-aggregate complex was found to form in single-stranded polycytidine chains, resulting in the appearance of a new absorption band at approximately 500 nm. The specific interaction of cytosine bases suggests the involvement of the C-rich i-motif in facilitating the formation of the H-aggregate complex. Second, the interaction of YOYO-3 with double-stranded poly­(A·T) tracts also led to the appearance of a new absorption band at approximately 500 nm, and hence of a different type of H-aggregate. We found that the aggregate is formed mainly in double-stranded regions with consecutive adenine bases in the same strand (and thymine bases in the complementary strand). These poly­(A·T) tracts provide narrow minor grooves and enhanced electrostatic negative potential to promote the aggregation of the negatively charged cyanine. As the YOYO-3 H-aggregates are nonfluorescent, our results provide an important basis to quantitatively understand the fluorescence emission of this cyanine dye in the presence of DNA strands

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 dye’s 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

Auswirkungen des Stabex-Systems auf die Stabilität der Exporterlöse - Eine empirische Analyse zum Nutzen partieller Stabilisierungselemente

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-(<i>o</i>-tolyl)­dibenzo­[<i>b</i>,<i>e</i>]­silin-3­(5<i>H</i>)-one (<b>2-Me TM</b>), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, p<i>K</i>_N‑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 <b>2-Me TM</b> 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 <i>pK</i>*_N‑A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether <b>2-Me TM</b> 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

Development of a New Dual Polarity and Viscosity Probe Based on the Foldamer Concept

Small molecular probes able to act as sensors are of enormous interest thanks to their multiple applications. Here, we report on the development of a novel supramolecular dual viscosity and polarity probe based on the foldamer concept, which increases the resolution limits of traditional probes at low viscosity values (0–4 mPa·s). The applicability of this new probe has been tested with a supramolecular organogel

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 (<b>2</b>, <b>6</b>) have bathochromically shifted absorption and fluorescence spectral maxima compared to those of the respective asymmetrically monosubstituted dyes (<b>1</b>, <b>5</b>). 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 <b>1</b>–<b>6</b> are high (0.7 ≤ Φ ≤ 1.0). The experimental results are backed up by quantum chemical calculations of the lowest electronic excitations in <b>1</b>, <b>2</b>, <b>5</b>, <b>6</b>, 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 <b>1</b>–<b>6</b> has been examined as a function of solvent by means of the recent, generalized treatment of the solvent effect, proposed by Catalán (<i>J. Phys. Chem. B</i> <b>2009</b>, <i>113</i>, 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

8‑HaloBODIPYs and Their 8‑(C, N, O, S) Substituted Analogues: Solvent Dependent UV–Vis Spectroscopy, Variable Temperature NMR, Crystal Structure Determination, and Quantum Chemical Calculations

The UV–vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the <i>meso</i>-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (<b>7</b>), 8-benzylamino (<b>8</b>), 8-methoxy (<b>9</b>), and 8-phenoxy (<b>10</b>) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)­BODIPY (<b>6</b>) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for <b>6</b>, <b>8</b>, and <b>10</b> have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8–N bond in <b>7</b> and <b>8</b> has been observed via temperature dependent ¹H NMR spectroscopy, whereas for <b>10</b> the rotation about the C8–O bond is not hindered. The crystal structure of <b>8</b> demonstrates that the short C8–N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of <b>10</b> shows a short C8–O bond and an intramolecular C–H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the <i>meso</i>-substituent on the spectroscopic properties