Alma R. Morales

Folate Receptor-Targeted Aggregation-Enhanced Near-IR Emitting Silica Nanoprobe for One-Photon in Vivo and Two-Photon ex Vivo Fluorescence Bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.

Resonant enhancement of two-photon absorption in substituted fluorene molecules

The degenerate and nondegenerate two-photon absorption (2PA) spectra for a symmetric and an asymmetric fluorene derivative were experimentally measured in order to determine the effect of intermediate state resonance enhancement (ISRE) on the 2PA cross section delta. The ability to tune the individual photon energies in the nondegenerate 2PA (ND-2PA) process afforded a quantitative study of the ISRE without modifying the chemical structure of the investigated chromophores. Both molecules exhibited resonant enhancement of the nonlinearity with the asymmetric compound showing as much as a twentyfold increase in delta. Furthermore, the possibility of achieving over a one order of magnitude enhancement of the nonlinearity reveals the potential benefits of utilizing ND-2PA for certain applications. To model ISRE, we have used correlated quantum-chemical methods together with the perturbative sum-over-states (SOS) expression. We find strong qualitative and quantitative correlation between the experimental and theoretical results. Finally, using a simplified three-level model for the SOS expression, we provide intuitive insight into the process of ISRE for ND-2PA.

Design, synthesis, and structural and spectroscopic studies of push-pull two-photon absorbing chromophores with acceptor groups of varying strength.

A new series of unsymmetrical diphenylaminofluorene-based chromophores with various strong π-electron acceptors were synthesized and fully characterized. The systematic alteration of the structural design facilitated the investigation of effects such as molecular symmetry and strength of electron-donating and/or -withdrawing termini have on optical nonlinearity. In order to determine the electronic and geometrical properties of the novel compounds, a thorough investigation was carried out by a combination of linear and nonlinear spectroscopic techniques, single-crystal X-ray diffraction, and quantum chemical calculations. Finally, on the basis of two-photon absorption (2PA) cross sections, the general trend for π-electron accepting ability, i.e., ability to accept charge transfer from diphenylamine was: 2-pyran-4-ylidene malononitrile (pyranone) > dicyanovinyl > bis(dicyanomethylidene)indane >1-(thiophen-2-yl)propenone > dicyanoethylenyl >3-(thiophen-2-yl)propenone. An analogue with the 2-pyran-4-ylidene malononitrile acceptor group exhibited a nearly 3-fold enhancement of the 2PA cross section (1650 GM at 840 nm), relative to other members of the series.

Fluorene-based metal-ion sensing probe with high sensitivity to Zn2+ and efficient two-photon absorption.

The photophysical, photochemical, two-photon absorption (2PA) and metal ion sensing properties of a new fluorene derivative (E)-1-(7-(4-(benzo[d]thiazol-2-yl)styryl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)-3-(2-(9,10,16,17,18,19,21,22,23,24-decahydro-6H dibenzo[h,s][1,4,7,11,14,17]trioxatriazacycloicosin-20(7H)-yl)ethyl)thiourea (1) were investigated in organic and aqueous media. High sensitivity and selectivity of 1 to Zn(2+) in tetrahydrofuran and a water/acetonitrile mixture were shown by both absorption and fluorescence titration. The observed complexation processes corresponded to 1:1 stoichiometry with the range of binding constants approximately (2-3) x 10(5) M(-1). The degenerate 2PA spectra of 1 and 1/Zn(2+) complex were obtained in the 640-900 nm spectral range with the maximum values of two-photon action cross section for ligand/metal complex approximately (90-130) GM, using a standard two-photon induced fluorescence methodology under femtosecond excitation. The nature of the 2PA bands was analyzed by quantum chemical methods and a specific dependence on metal ion binding processes was shown. Ratiometric fluorescence detection (420/650 nm) provided a good dynamic range (10(-4) to 10(-6) M) for detecting Zn(2+), which along with the good photostability and 2PA properties of probe 1 makes it a good candidate in two-photon fluorescence microscopy imaging and sensing of Zn ions.

Amine-Reactive Fluorene Probes: Synthesis, Optical Characterization, Bioconjugation, and Two-Photon Fluorescence Imaging

With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially “lighting up” after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.

Biomolecule labeling and imaging with a new fluorenyl two-photon fluorescent probe.

Closely involved in the progression of nonlinear bioimaging is the development of optical probes for investigating biological function and activity. Introduction of new fluorescent compounds possessing enhanced nonlinearities is essential for advancing the utility of two-photon absorption (2PA) processes in the biological sciences. Herein, we report the synthesis of fluorene-based fluorophores tailored for multiphoton imaging, incorporating the succinimidyl ester and thioester functionality as reactive linkers for further coupling with a wide variety of biologically relevant molecules. The succinimidyl ester amine reactive probe was conjugated with the cyclic peptide RGDfK and polyclonal antirat IgG protein. Upon conjugation, the basic molecular architecture and photophysical properties of the active 2PA chromophore remain unchanged. Conventional and two-photon fluorescence microscopy (2PFM) imaging of COS-7 and HeLa cells, incubated with either the fluorene-RGD peptide conjugate or the fluorene-IgG conjugate, was demonstrated. The fluorene-IgG conjugate was used to image cell spindles at early mitotic developmental stages.

Synthesis and Characterization of New Fluorene-Based Singlet Oxygen Sensitizers

The synthesis, photophysical characterization, and determination of singlet oxygen quantum yields (ΦΔ) for a class of fluorene derivatives with potential application in two-photon photodynamic therapy (PDT) is reported. It has been demonstrated that these compounds possess the ability to generate singlet oxygen (1O2) upon excitation. A photochemical method, using 1,3-diphenylisobenzofuran (DPBF) as 1O2 chemical quencher, was employed to determine the singlet oxygen quantum yields (ΦΔ) of the fluorene-based photosensitizers in ethanol. ΦΔ values ranged from 0.35 to 0.75. These derivatives may have potential application as two-photon photosensitizers when pumped via two-photon excitation in the near-IR spectral region.

Integrin-Targeting Block Copolymer Probes for Two-Photon Fluorescence Bioimaging

Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for noninvasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl) and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cell viability and 2PFM imaging of human epithelial U87MG cell lines that overexpress α(v)β(3) integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin-rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin-targeting ability of the new probes.

One- and two-photon fluorescence anisotropy of selected fluorene derivatives.

The steady-state excitation anisotropy spectra of fluorene derivatives were measured in viscous solvents, under the one- and two-photon excitation, over a broad spectral range (UV–Visible). The orientation of their absorption transition moments for the first, S0→S1, and second, S0→S2, excited states were determined. It was shown experimentally that a decrease in the angle between S0→S1and S0→S2 transitions corresponded to an increased value of two-photon absorption (2PA) cross section for these molecules. Two-photon excitation anisotropy was nearly constant over the spectral region investigated (in contrast to one-photon excitation anisotropy spectra) and can be roughly explained by a simple model of 2PA based on the single intermediate state approximation. For comparison, the same trend in two-photon excitation anisotropy was observed for Rhodamine B inglycerol.

Steady-state and femtosecond transient absorption spectroscopy of new two-photon absorbing fluorene-containing quinolizinium cation membrane probes.

The synthesis, linear photophysical characterization, and nonlinear optical properties of two new symmetrical fluorene-containing quinolizinium derivatives, 2,8-bis((E)-2-(7-(diphenylamino)-9,9-dihexyl-9H-fluoren-2-yl)vinyl)quinolizinium hexafluorophosphate (1) and 2,8-bis((E)-2-(7-((7-(diphenylamino)-9,9-dihexyl-9H-fluoren-2-yl)ethynyl)-9,9-dihexyl-9H-fluoren-2yl)vinyl)quinolizinium hexafluorophosphate (2), are reported. The nature of the dual-band steady-state fluorescence emission of 1 and 2 was determined, and violation of Kashas rule along with a strong dependence on solvent polarity were shown. A relatively complex structure of two-photon absorption (2PA) spectra of 1 and 2, with maximum cross sections of ∼400-600 GM, was determined using the open aperture Z-scan method. Different types of fast relaxation processes with characteristic times of 0.3-0.5 ps and 1.5-2 ps were observed in the excited states of the new compounds via femtosecond transient absorption pump-probe spectroscopy. To better understand the photophysical behavior of 1 and 2, a quantum-mechanical study was undertaken using TD-DFT and ZINDO/S methods. Simulated linear absorption spectra were found to be in good agreement with experimental data, while 2PA cross sections were overestimated. Although the new dyes were highly fluorescent in nonpolar solvents, they were essentially nonfluorescent in polar media. Significantly, the quinolizinium dyes exhibited fluorescence turn-on behavior upon binding to bovine serum album (BSA) protein, exhibiting over 4-fold fluorescence enhancement, which was a finding that was leveraged to demonstrate cell membrane fluorescence imaging of HeLa cells.