Two-Color Two-Photon Excitation of Fluorescence

Abstract— We report the observation of two-photon excitation of an organic fluorophore with two different wavelengths, a phenomenon we refer to as two-color two-photon (2C2P) excitation. Ultraviolet emission of p -Merphenyl at 340 nm was observed when the sample was illuminated with both 375 and 750 nm pulses from a picosecond dye laser. The emission of p -terphenyl was about 100-fold and more than 1000-fold less for illumination at only 375 or 750 nm, respectively. Observation of the 2C2P signal required temporal and spatial overlap of the 375 and 750 nm pulses. The amplitude of the signal depended on the polarization of each beam. 2C2P excitation can have applications in fluorescence microscopy and elsewhere when spatially localized excitation is desirable.     

RAPID COMMUNICATION

Abstract— We show that the calcium fiuorophore Indo-1 can be excited by simultaneous absorption of three-photons at 885 nra, a wavelength readily available from Ti:sapphire lasers. Three-photon excitation was demonstrated by the emission intensity of Indo-1 which depended on the cube of the laser power, and by a higher anisotropy than was observed for two-photon excitation. Excitation of Indo-1 becomes a two-photon process when the wavelength is decreased to 820 nm. Three-photon excitation was accomplished at a low 17μ concentration of Indo-1. Examination of the spatial profile of the excited Indo-1 showed a smaller volume for three- versus two-photon excitation. These results suggest that three-photon excitation may be useful in fluorescence microscopy using the long wavelength output of Tksapphire lasers, and may provide higher spatial resolution than available using two-photon excitation.     

Distance-dependent fluorescence quenching ofN-acetyl-l-tryptophanamide by acrylamide

We examined the time-dependent intensity decays ofN-acetyl-l-tryptophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol over a range of temperatures. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature, and quenching of NATA by acrylamide was observed even in vitrified propylene glycol, where translational diffusion cannot occur during the lifetime of the excited state. These frequencydomain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide, and the results are consistent with a rate constant for quenching that depends exponentially on the fluorophore-quencher separation distance. The exponential distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, and the steady-state data aid in determining the interaction distance between NATA and acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of acrylamide quenching of proteins.     

Coordination Sites for Sodium and Potassium Ions in Nucleophilic Adeninate Contact ion-Pairs: A Molecular-Wide and Electron Density-Based (MOWED) Perspective

The adeninate anion (Ade⁻) is a useful nucleophile used in the synthesis of many prodrugs (including those for HIV AIDS treatment). It exists as a contact ion-pair (CIP) with Na⁺ and K⁺ (M⁺) but the site of coordination is not obvious from spectroscopic data. Herein, a molecular-wide and electron density-based (MOWED) computational approach implemented in the implicit solvation model showed a strong preference for bidentate ion coordination at the N3 and N9 atoms. The N3N9-CIP has (i) the strongest inter-ionic interaction, by −30 kcal mol⁻¹, with a significant (10–15%) covalent contribution, (ii) the most stabilized bonding framework for Ade⁻, and (iii) displays the largest ion-induced polarization of Ade⁻, rendering the N3 and N9 the most negative and, hence, most nucleophilic atoms. Alkylation of the adeninate anion at these two positions can therefore be readily explained when the metal coordinated complex is considered as the nucleophile. The addition of explicit DMSO solvent molecules did not change the trend in most nucleophilic N-atoms of Ade⁻ for the in-plane M-Ade complexes in M-Ade-(DMSO)₄ molecular systems. MOWED-based studies of the strength and nature of interactions between DMSO solvent molecules and counter ions and Ade⁻ revealed an interesting and unexpected chemistry of intermolecular chemical bonding.     

Highly Z-Selective Horner–Wadsworth–Emmons Olefination Using Modified Still–Gennari-Type Reagents

In this report, new, easily accessible reagents for highly Z-selective HWE reactions are presented. Alkyl di-(1,1,1,3,3,3-hexafluoroisopropyl)phosphonoacetates, structurally similar to Still–Gennari type reagents, were tested in HWE reactions with a series of various aldehydes. Very good Z-selectivity (up to a 98:2 Z:E ratio) was achieved in most cases along with high yields. Application of the new reagents may be a valuable, practical alternative to the well-established Still–Gennari or Ando Z-selective carbonyl group olefination protocols.     

QUENCHING OF METHYLCYCLOHEXANE FLUORESCENCE BY METHANOL*

We measured the fluorescence emission spectrum and intensity decays of methylcyclohexane (MCH) when excited by simultaneous absorption of two photons at 298 nm. The steady-state intensities and lifetimes were both decreased by methanol, which was found to be an efficient quencher of MCH fluorescence. Methanol quenching of MCH is clearly dynamic, but the exact mechanism of quenching is unclear. Dynamic quenching of MCH was also observed by water and n-propanol. These results suggest that alkane fluorescence from biopolymers, if observable, will only occur from regions of the macromolecules that are not exposed to water.     

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.     

A Molecular-Wide and Electron Density-Based Approach in Exploring Chemical Reactivity and Explicit Dimethyl Sulfoxide (DMSO) Solvent Molecule Effects in the Proline Catalyzed Aldol Reaction

Modelling of the proline (1) catalyzed aldol reaction (with acetone 2) in the presence of an explicit molecule of dimethyl sulfoxide (DMSO) (3) has showed that 3 is a major player in the aldol reaction as it plays a double role. Through strong interactions with 1 and acetone 2, it leads to a significant increase of energy barriers at transition states (TS) for the lowest energy conformer 1a of proline. Just the opposite holds for the higher energy conformer 1b. Both the ‘inhibitor’ and ‘catalyst’ mode of activity of DMSO eliminates 1a as a catalyst at the very beginning of the process and promotes the chemical reactivity, hence catalytic ability of 1b. Modelling using a Molecular-Wide and Electron Density-based concept of Chemical Bonding (MOWED-CB) and the Reaction Energy Profile–Fragment Attributed Molecular System Energy Change (REP-FAMSEC) protocol has shown that, due to strong intermolecular interactions, the HN-C-COOH (of 1), CO (of 2), and SO (of 3) fragments drive a chemical change throughout the catalytic reaction. We strongly advocate exploring the pre-organization of molecules from initially formed complexes, through local minima to the best structures suited for a catalytic process. In this regard, a unique combination of MOWED-CB with REP-FAMSEC provides an invaluable insight on the potential success of a catalytic process, or reaction mechanism in general. The protocol reported herein is suitable for explaining classical reaction energy profiles computed for many synthetic processes.