Analysis of the autocorrelation functions Examples of normalized autocorrelation curves obtained for EGFP at pH=5 for different temperatures are shown in Fig. I.

No abstract

SummaryFluorescence excitation can result in the formation of reactive oxygen species and free radicals damaging to live cells. In the case of erythrocytes, reaction of these reactive oxygen species with membrane components causes large-scale morphological changes followed by cell haemolysis. In an effort to understand the origin of these morphological changes, we have studied the consequences of localized photodamage on the erythrocyte membrane. For this, we irradiated a small area of the cell membrane using a focused laser beam in the presence of an external photosensitizer. We observed the rapid formation of an invagination (approximately 1 μm deep) at the laser focus, long before photohaemolysis occurred. We measured the rate of invagination formation and the rate of cell haemolysis, using a combination of fluorescence contrast imaging (to detect the membrane position) with fluorescence correlation spectroscopy (to measure photosensitizer concentration). We found that the kinetics of both processes depend in a similar manner on light energy flux, fluorophore concentration and the presence of oxygen scavenger. This leads us to the conclusion that the observed invagination is due to the photooxidation of membrane-associated proteins, representing a precursor of cellular photohaemolysis. We then discuss two different molecular mechanisms (conformational change of the protein band 3 and detachment of the spectrin cytoskeleton from the lipid membrane) that may explain how the photodamage of membraneassociated proteins can lead to a deformation of the lipid bilayer.

Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones, with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and x-ray crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence lifetimes are less than 1 ns, suggesting that there may be a significant energy transfer to the triplet state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cytotoxicity studies with a number of human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that increasing the number of double bonds, from one to two, in the piperidone ''wings'' increases the photo-toxicity with little corresponding change in the dark cyto-toxicity. Two-photon photo-toxicity studies are also underway (exposure in the range of 740 -860 nm) as well as singlet oxygen detection studies(detection at about 1270 nm).