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

Abstract

Singlet molecular oxygen, O 2 (a 1 ∆ g ), can be created in a single cell from ground state oxygen, O 2 (X 3 Σ g -), upon focused laser irradiation of an intracellular sensitizer. This cytotoxic species can subsequently be detected by its 1270 nm phosphorescence (a 1 ∆ g → X 3 Σ 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) and, as such, are 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.3

Simultaneous FRAP, FLIM and FAIM for measurements of protein mobility and interaction in living cells

“…Time-resolved fluorescence anisotropy imaging (tr-FAIM) [17,[25][26][27] is currently rarely used in biology but has potential to be a powerful indicator of rotational diffusion of fluorophores, protein structure or function [1]. Underpinning tr-FAIM, fluorescence lifetime imaging microscopy (FLIM) [2,3,[28][29][30][31][32][33] maps the fluorescence lifetime in every pixel of an image and is a powerful technique for probing the local environment of a fluorophore as the measured lifetime is largely independent of fluorophore concentration, but can be sensitive to pH [34], refractive index [35][36][37][38], reactive quenching species [39] and viscosity [30,31,[40][41][42]. Both fluorescence anisotropy and FRAP have previously been used independently for a study of aggregation states of alpha-synuclein, a protein which plays a role in Parkinson's disease [43], and an arrangement for dynamic FRAP and rotational diffusion measurements for colloids has been presented [44].…”

Tuning the Sensitivity of Fluorescent Porphyrin Dimers to Viscosity and Temperature

“…The excitation spectra (Figure S1 in the Supporting Information) reveal distinct wavelength regions in which pure “planar” and pure “twisted” excited state conformers can be excited, with excitation maxima independent of the solvent viscosity 24. While only a small overlap in twisted and planar excitation spectra was recorded for 1 , 2 and 4, the spectra for 3 show a significant overlap (see Figure S1).…”

Luminescence spectroscopy of singlet oxygen enables monitoring of oxygen consumption in biological systems consisting of fatty acids

“…This change in local oxygen concentration causes a gradient of oxygen concentration that should be balanced by oxygen diffusion. 10,37 The small diffusion coefficients for oxygen in solvents may hamper the diffusion-controlled process of 37 at 303 K and the laser spot size in the cuvette is 8 mm.…”