Kevin D. Belfield

Multiphoton-absorbing organic materials for microfabrication, emerging optical applications and non-destructive three-dimensional imaging

Non-resonant two-photon absorption (TPA) can be defined as the simultaneous absorption of two photons, via a virtual state, in a medium. TPA exhibits a quadratic dependence of absorption on the incident light intensity, resulting in highly localized photoexcitation. Recent developments in the design and synthesis of efficient, stable TPA organic materials are discussed. Microfabrication via two-photon induced free radical polymerization of acrylate monomers and cationic polymerization of epoxide monomers was accomplished using commercially available photoinitiators, and also a custom-made compound possessing high two-photon absorptivity. Two-photon facilitated photoisomerization of a fulgide in solution and in a polymer thin film demonstrated two-photon induced photochromism and its application in interferometric image recording, respectively. Greatly enhanced signal-to-noise ratios and resolution were achieved in the non-destructive three-dimensional two-photon fluorescence imaging of a polymer-coated substrate versus conventional single-photon laser scanning confocal microscopic imaging. Multifunctional TPA organic materials and fabrication of functional microstructures are also discussed. Copyright

High-Fidelity Hydrophilic Probe for Two-Photon Fluorescence Lysosomal Imaging

The synthesis and characterization of a novel two-photon-absorbing fluorene derivative, LT1, selective for the lysosomes of HCT 116 cancer cells, is reported. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for two-photon fluorescence microscopy (2PFM) lysosomal imaging. The cytotoxicity of the probe was investigated to evaluate the potential of using this probe for live two-photon fluorescence biological imaging applications. Colocalization studies of the probe with commercial Lysotracker Red in HCT 116 cells demonstrated the specific localization of the probe in the lysosomes with an extremely high colocalization coefficient (0.96). A figure of merit was introduced to allow comparison between probes. LT1 has a number of properties that far exceed those of commercial lysotracker probes, including higher two-photon absorption cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. 2PFM was used to demonstrate lysosomal tracking with LT1.

Two-photon absorption cross-sections of common photoinitiators

Recent interests in and applications of two-photon absorption (2PA) induced photopolymerization have afforded advanced opportunities to perform three-dimensionally resolved polymerization, resulting in intricate microfabrication and imaging. Many of the reported 2PA-induced polymerizations make use of commercially available photoinitiators, and a key parameter to consider is the two-photon absorption cross-section (δ) of the initiator. To date, there has been no comprehensive investigation of two-photon absorptivity of commercial photoinitiators, though a few studies presenting novel photoinitiators for two-photon polymerization have appeared. Herein, we report the 2PA properties of common, commercially available photoinitiators typically utilized in conventional radiation curing science and technologies, and often used in 2PA-based polymerizations. Z-scan and white-light continuum (WLC) pump–probe techniques were utilized to obtain two-photon absorption cross-sections ( δ). The results for most compounds were found to yield good agreement between the two methods. Most of the photoinitiators studied possess low δ, except Irgacure OXE01, indicating a need for the development of new photoinitiators with improved properties optimized for 2PA applications. A compound prepared in our laboratories exhibits high 2PA and was useful as a two-photon free-radical photoinitiator.

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.

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides.

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore’s usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H2O2-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially “light up” in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.

Highly Selective Fluorescence Turn-On Sensor for Fluoride Detection

Through click chemistry, triazole and triazolium groups have been explored to recognize anions through C-H···A(-) hydrogen-bonding complexion. Herein, we demonstrate evidence of fluoride-induced deprotonation of a C-H bond and its application in fluoride detection. The combination of fluorene and triazolium units produced a highly selective fluorescence turn-on prototype sensor for fluoride. The interactions between the C-H bond and F(-) were studied by fluorescence spectroscopy and (1)H NMR titrations. Test papers were prepared to detect fluoride in aqueous media at concentrations down to 1.9 ppm, important for estimating whether the fluoride concentration in drinking water is at a safe level.

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.

Donor−Acceptor−Donor Fluorene Derivatives for Two-Photon Fluorescence Lysosomal Imaging

As part of a strategy to achieve large two-photon absorptivity in fluorene-based probes, a series of donor-acceptor-donor (D-A-D) type derivatives were synthesized and their two-photon absorption (2PA) properties investigated. The synthesis of D-A-D fluorophores was achieved by efficient preparation of key intermediates for the introduction of central electron acceptor groups. To accomplish the synthesis of two of the new derivatives, a high-yield method for a one-step direct dibromomethylation of phenyl sulfide was developed. The linear and nonlinear optical properties, including UV-vis absorption, fluorescence emission, fluorescence anisotropy, and two-photon absorption (2PA), of the new D-A-D compounds were measured and compared to their D-A or D-D counterparts. Fully conjugated acceptor moieties in the center of the D-A-D fluorophore led to the greatest increase in the 2PA cross section, while weakly conjugated central acceptors exhibited only a modest increase in the 2PA cross section relative to D-A diploar analogs. Encapsulation of the new probes in Pluronic F 108NF micelles, and subsequent incubation in HCT 116 cells, resulted in very high lysosomal colocalization (>0.98 colocalization coefficient) relative to commercial Lysotracker Red, making the micelle-encapsulated dyes particularly attractive as fluorescent probes for two-photon fluorescence microscopy lysosomal imaging.

Novel BODIPY-Based Fluorescence Turn-on Sensor for Fe3+ and Its Bioimaging Application in Living Cells

A novel boron-dipyrromethene (BODIPY) based fluorescence turn-on sensor for detecting Fe(3+) in aqueous media is reported with 23-fold fluorescence enhancement. The sensor is comprised of a combination of BODIPY fluorophore and a new Fe(3+)-recognizing cryptand that exhibits high selectivity, sensitivity, and reversibility toward Fe(3+) detection. Cell imaging studies demonstrate that this sensor is capable of sensing Fe(3+) in living cells.

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.