Peter R. Ogilby

Imaging intracellular viscosity of a single cell during photoinduced cell death.

Diffusion-mediated cellular processes, such as metabolism, signalling and transport, depend on the hydrodynamic properties of the intracellular matrix. Photodynamic therapy, used in the treatment of cancer, relies on the generation of short-lived cytotoxic agents within a cell on irradiation of a drug. The efficacy of this treatment depends on the viscosity of the medium through which the cytotoxic agent must diffuse. Here, spectrally resolved fluorescence measurements of a porphyrin-dimer-based molecular rotor are used to quantify intracellular viscosity changes in single cells. We show that there is a dramatic increase in the viscosity of the immediate environment of the rotor on photoinduced cell death. The effect of this viscosity increase is observed directly in the diffusion-dependent kinetics of the photosensitized formation and decay of a key cytotoxic agent, singlet molecular oxygen. Using these tools, we provide insight into the dynamics of diffusion in cells, which is pertinent to drug delivery, cell signalling and intracellular mass transport.

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.

Singlet Oxygen Sensor Green®: Photochemical Behavior in Solution and in a Mammalian Cell

The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green® (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation‐dependent adverse effects in the system being investigated.

Control and Selectivity of Photosensitized Singlet Oxygen Production: Challenges in Complex Biological Systems

Singlet molecular oxygen is a reactive oxygen species that plays an important role in a number of biological processes, both as a signalling agent and as an intermediate involved in oxidative degradation reactions. Singlet oxygen is commonly generated by the so‐called photosensitization process wherein a light‐absorbing molecule, the sensitizer, transfers its energy of excitation to ground‐state oxygen to make singlet oxygen. This process forms the basis of photodynamic therapy, for example, where light, a sensitizer, and oxygen are used to initiate cell death and ultimately destroy undesired tissue. Although the photosensitized production of singlet oxygen has been studied and used in biologically pertinent systems for years, the photoinitiated behaviour is often indiscriminate and difficult to control. In this Concept, we discuss new ideas and results in which spatial and temporal control of photosensitized singlet oxygen production can be implemented through the incorporation of the sensitizer into a conjugate system that selectively responds to certain triggers or stimuli.

Time-resolved Singlet Oxygen Phosphorescence Measurements from Photosensitized Experiments in Single Cells: Effects of Oxygen Diffusion and Oxygen Concentration

Time‐resolved singlet oxygen, O2(a1Δg), phosphorescence experiments have been performed in single cells upon pulsed laser irradiation of a photosensitizer incorporated into the cell. Data recorded as a function of the partial pressure of ambient oxygen to which the cell is exposed reflect apparent values for the intracellular oxygen diffusion coefficient and intracellular oxygen concentration that are smaller than those found in neat H2O. This conclusion is supported by O2(a1Δg) phosphorescence data and sensitizer triplet state absorption data recorded in control experiments on sucrose solutions with different viscosities. We recently demonstrated that the intracellular lifetime of O2(a1Δg) is comparatively long (∼3 μs) and does not differ significantly from that in neat H2O (∼3.5 μs). Despite this long lifetime, however, our estimate of an apparent intracellular oxygen diffusion coefficient in the range ∼2–4 × 10−6 cm2 s−1 means that the spatial domain of intracellular O2(a1Δg) activity will likely have a spherical radius of ∼100 nm. This latter point helps reconcile seeming inconsistencies between our direct O2(a1Δg) lifetime data and results obtained from independent photobleaching experiments that show a limited translational diffusion distance for O2(a1Δg) within a cell.

Overview of Theoretical and Computational Methods Applied to the Oxygen-Organic Molecule Photosystem

Abstract The challenges of using modern theoretical and computational tools to model the unique features of the oxygen–organic molecule photosystem are discussed from a historical and pedagogical perspective. This review is written for the novice, but the problems formulated should stimulate the expert.

Reaction of Singlet Oxygen with Tryptophan in Proteins: A Pronounced Effect of the Local Environment on the Reaction Rate

Singlet molecular oxygen, O(2)(a(1)Δ(g)), can influence many processes pertinent to the function of biological systems, including events that result in cell death. Many of these processes involve a reaction between singlet oxygen and a given amino acid in a protein. As a result, the behavior of that protein can change, either because of a structural alteration and/or a direct modification of an active site. Surprisingly, however, little is known about rate constants for reactions between singlet oxygen and amino acids when the latter are in a protein. In this report, we demonstrate using five separate proteins, each containing only a single tryptophan residue, that the rate constant for singlet oxygen reaction with tryptophan depends significantly on the position of this amino acid in the protein. Most importantly, the reaction rate constant depends not only on the accessibility of the tryptophan residue to oxygen, but also on factors that characterize the local molecular environment of the tryptophan in the protein. The fact that the local protein environment can either appreciably inhibit or accelerate the reaction of singlet oxygen with a given amino acid can have significant ramifications for singlet-oxygen-mediated events that perturb cell function.

Oxygen-dependent photochemistry and photophysics of "miniSOG," a protein-encased flavin.

Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so‐called “mini Singlet Oxygen Generator” (miniSOG), an FMN‐containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron‐transfer reactions (Type‐I chemistry) at the expense of photosensitized singlet oxygen production (Type‐II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well‐characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.

5,10,15,20-Tetrakis(N-Methyl-4-Pyridyl)-21H,23H-Porphine (TMPyP) as a Sensitizer for Singlet Oxygen Imaging in Cells: Characterizing the Irradiation-dependent Behavior of TMPyP in a Single Cell†

Abstract Singlet molecular oxygen, a1Δg, can be detected from a single cell by its weak 1270 nm phosphorescence (a1Δg→X3Σg−) upon irradiation of the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) incorporated into the cell. The behavior of this sensitizer in a cell, and hence the behavior of the associated singlet oxygen phosphorescence signal, depends on the conditions under which the sample is exposed to light. Upon irradiation of a neuron freshly incubated with TMPyP, the intensity of TMPyP fluorescence initially increases and there is a concomitant increase in the singlet oxygen phosphorescence intensity from the cell. These results appear to reflect a photoinduced release of TMPyP bound to DNA in the nucleus of the cell, where TMPyP tends to localize, and the subsequent relocalization of TMPyP to a different microenvironment in the cell. Upon prolonged irradiation of the cell, TMPyP photobleaches and there is a corresponding decrease in the singlet oxygen phosphorescence intensity from the cell. The data reported herein provide insight into key factors that can influence photosensitized singlet oxygen experiments performed on biological samples.

Aarhus Sensor Green: A Fluorescent Probe for Singlet Oxygen

A tetrafluoro-substituted fluorescein derivative covalently linked to a 9,10-diphenyl anthracene moiety has been synthesized, and its photophysical properties have been characterized. This compound, denoted Aarhus Sensor Green (ASG), has distinct advantages for use as a fluorescent probe for singlet molecular oxygen, O2(a(1)Δg). In the least, ASG overcomes several limitations inherent to the use of the related commercially available product called Singlet Oxygen Sensor Green (SOSG). The functional behavior of both ASG and SOSG derives from the fact that these weakly fluorescent compounds rapidly react with singlet oxygen via a π2 + π4 cycloaddition to irreversibly yield a highly fluorescent endoperoxide. The principal advantage of ASG over SOSG is that, at physiological pH values, both ASG and the ASG endoperoxide (ASG-EP) do not themselves photosensitize the production of singlet oxygen. As such, ASG better fits the requirement of being a benign probe. Although ASG readily enters a mammalian cell (i.e., HeLa) and responds to the presence of intracellular singlet oxygen, its behavior in this arguably complicated environment requires further investigation.