Gokhan Yahioglu

Singlet Oxygen in a Cell: Spatially Dependent Lifetimes and Quenching Rate Constants

Singlet molecular oxygen, O(2)(a(1)Delta(g)), can be created in a single cell from ground-state oxygen, O(2)(X(3)Sigma(g)(-)), upon focused laser irradiation of an intracellular sensitizer. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a(1)Delta(g) --> X(3)Sigma(g)(-)) with subcellular spatial resolution. The singlet oxygen lifetime determines its diffusion distance and hence the intracellular volume element in which singlet-oxygen-initiated perturbation of the cell occurs. In this study, the time-resolved phosphorescence of singlet oxygen produced by the sensitizers chlorin (Chl) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) was monitored. These molecules localize in different domains of a living cell. The data indicate that (i) the singlet oxygen lifetime and (ii) the rate constant for singlet oxygen quenching by added NaN(3) depend on whether Chl or TMPyP was the photosensitizer. These observations likely reflect differences in the chemical and physical constituency of a given subcellular domain (e.g., spatially dependent oxygen and NaN(3) diffusion coefficients), thereby providing evidence that singlet oxygen responds to the inherent heterogeneity of a cell. Thus, despite a relatively long intracellular lifetime, singlet oxygen does not diffuse a great distance from its site of production. This is a consequence of an apparent intracellular viscosity that is comparatively large.

THIRD-ORDER OPTICAL NON-LINEARITY IN ZN(II) COMPLEXES OF 5,10,15,20-TETRAARYLETHYNYL-SUBSTITUTED PORPHYRINS

Abstract Third-order optical nonlinearity χ(3) of complexes in 5,10,15,20-tetraarylethynyl-substituted porphyrins has been measured using the Z-scan technique. The real and imaginary parts of χ(3) were measured at three different wavelengths and found to be in the region 1.05 × 10−12 esu and 5.2 × 10−12 esu, respectively. Experimental characterisation shows a saturation absorption at wavelengths close to resonance and reverse saturation absorption at off-resonance wavelengths.

Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments

Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug‐coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide‐a and clinically approved, verteporfin photosensitizers) to single‐chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti‐HER‐2 scFv linked to 8–10 molecules of pyropheophorbide‐a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer.

Molecular rheometry: Direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

Fluorescence Anisotropy of Molecular Rotors

We present polarization-resolved fluorescence measurements of fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ), 9-(2,2-dicyanovinyl)julolidine (DCVJ), and a meso-substituted boron dipyrromethene (BODIPY-C(12)). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore. Herein we evaluate the applicability of molecular rotors as probes of apparent viscosity on a microscopic scale based on their viscosity dependent fluorescence depolarization. We develop a theoretical framework, combining the Förster-Hoffmann equation with the Perrin equation and compare the dynamic ranges and usable working regimes for these dyes in terms of utilising fluorescence anisotropy as a measure of viscosity. We present polarization-resolved fluorescence spectra and steady-state fluorescence anisotropy imaging data for measurements of intracellular viscosity. We find that the dynamic range for fluorescence anisotropy for CCVJ and DCVJ is significantly lower than that of BODIPY-C(12) in the viscosity range 0.6<η<600 cP. Moreover, using steady-state anisotropy measurements to probe microviscosity in the low (<3 cP) viscosity regime, the molecular rotors can offer a better dynamic range in anisotropy compared with a rigid dye as a probe of microviscosity, and a higher total working dynamic range in terms of viscosity.

Pulsed excitation of low-mobility light-emitting diodes: Implication for organic lasers

We present a theoretical and experimental study of electrical pulsed excitation in polymer light-emitting diodes (LEDs). We find that the low mobility results in a relatively high charge density within an electrically pumped structure. The broad spectrum of the charge-induced absorption and its overlap with the ground-state absorption pose a significant barrier for achieving net gain and lasing. We suggest an approach that might circumvent this problem using suitable emitters and an appropriate driving scheme. We also suggest methods for ultrafast modulation of LEDs as well as direct determination of the space charge within the recombination zone.

Monitoring sol-to-gel transitions via fluorescence lifetime determination using viscosity sensitive fluorescent probes.

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials.

Properties of light emitting organic materials within the context of future electrically pumped lasers

The quest for electrically pumped organic laser is now conducted in several places around the globe. In this paper we outline our approach to this long-term project and aim to describe both encouraging and challenging (discouraging?) results. We describe experimental results using polymers, polymer blends and triplet emitters in an LED configuration using either CW or pulsed mode of operation. We also describe results of modeling such structures with the aim of being able to design electrical and optical characteristics of future laser structures. Finally we describe an approach to modify material parameters so as to provide a range of building blocks for optoelectronic devices.

Fluorescence characterisation of multiply-loaded anti-HER2 single chain Fv-photosesitizer conjugates suitable for photodynamic therapy

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (<or=20%) from non-covalently bound molecules. Confocal fluorescence microscopy demonstrates good cellular uptake of PPa conjugate with the HER2-positive cell line, SKOV-3, while negligible cell uptake is demonstrated for the HER2-negative cell line, KB. For the VP conjugate, increased rate of cellular uptake and prolonged retention in SKOV-3 cells is observed compared to free photosensitizer. In clinical applications this could provide increased potency and desired selectivity towards malignant tissue, leaving surrounding healthy tissue unharmed and reducing skin photosensitivity. The present study highlights the usefulness of photosensitizer immunoconjugates with scFvs for targeted PDT.

Tuning the red emission of a soluble poly(p-phenylene vinylene) upon grafting of porphyrin side groups

Abstract We investigate the energy transfer in donor–acceptor systems with reduced spectral overlap to assess the scope for emission colour tunability and the influence of spectral overlap on the luminescence efficiency. We use copolymers derived from poly [2-methoxy, 5- (2′-ethylhexyloxy)-1,4-phenylene vinylene], randomly attaching tetraphenylporphyrin side-chain units. We find that both photoluminescence and electroluminescence are widely tunable via control of the porphyrin concentration, with relatively efficient energy transfer in spite of limited spectral overlap. We also find that luminescence efficiency is controlled by concentration quenching of the tetraphenylporphyrin, with only a minor effect of spectral overlap. This has implications for electrophosphorescent devices.