Localized photodamage of the human erythrocyte membrane causes an invagination as a precursor of photohaemolysis

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.

Contractile dynamics change before morphological cues during fluorescence illumination

“…Morphology is a common indicator of cell viability. Evaluating morphological changes during light exposure to assess possible cytotoxicity is a common practice used widely across various experimental studies 4 5 6 7 8 9 10 11 12 13 14 . When affected by light, cells often exhibit morphologies such as: necrosis, blebbing, formation of multiple nuclei or vacuoles, and swelling of mitochondria 3 9 .…”

Widefield standing wave microscopy of red blood cell membrane morphology with high temporal resolution

“…We also investigated whether the SW technique caused any further photo-toxicity to the specimen compared to widefield epifluorescence imaging. It was observed that when using either imaging method, the red blood cell appeared to decay at a similar rate and in a manner that resembled the decay observed previously over 12 minutes under periodic focused laser illumination [ 62 ]. In the study of Wong et.…”

“…In the study of Wong et. al [ 62 ], it was found that membrane damage occurred even at low laser powers as the result of reactive oxygen species being generated via photo-hemolysis. This process has been hypothesized to be due to interactions between the reactive oxygen species and the membrane proteins, band 3 and spectrin [ 62 ], which has also been reported in studies using widefield illumination [ 63 , 64 ].…”

“…al [ 62 ], it was found that membrane damage occurred even at low laser powers as the result of reactive oxygen species being generated via photo-hemolysis. This process has been hypothesized to be due to interactions between the reactive oxygen species and the membrane proteins, band 3 and spectrin [ 62 ], which has also been reported in studies using widefield illumination [ 63 , 64 ]. It has also been found that when using multiphoton microscopy, red blood cell photodamage could be observed on timescales in the region of tens of seconds [ 65 ], however, when we carried out our video-rate experiments we observed no photo-toxicity effects over the 33 second duration.…”