Short and long-term phototoxicity in cells expressing genetic reporters under nanosecond laser exposure.
Nanosecond-duration laser pulses are exploited in a plethora of therapeutic and diagnostic applications, such as optoacoustic imaging. However, phototoxicity effects of pulsed radiation in living cells, in particular those expressing genetic reporters, are not well understood. We established a three-dimensional fluorescent protein expressing cellular model in order to reliably investigate the extent and major exposure parameters responsible for both photobleaching and phototoxicity under pulsed laser exposure, unveiling a variety of possible effects on living cells, from reversible photobleaching to cytotoxicity and cell death. Significant losses of fluorescence levels were identified when exposing the cells to illumination conditions considered safe under common standards for skin exposure in diagnostic imaging applications. Thus, the use of photolabile fluorescent proteins and their in vivo exposure parameters have to be designed carefully for all applications using pulsed nanosecond radiation. In particular, loss of signal due to bleaching may significantly alter signals in longitudinal measurements, making data quantification challenging.
Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg 85764, Germany.
Sven
S
Gottschalk
Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg 85764, Germany.
Héctor
H
Estrada
Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg 85764, Germany.
Oleksiy
O
Degtyaruk
Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg 85764, Germany; Faculty of Medicine, Technische Universität München, München 81675, Germany.
Johannes
J
Rebling
Institute of Radiation Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany.
Olena
O
Klymenko
Institute of Radiation Biology, Helmholtz Zentrum München, Neuherberg 85764, Germany.
Michael
M
Rosemann
Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg 85764, Germany; Faculty of Medicine, Technische Universität München, München 81675, Germany. Electronic address: dr@tum.de.
Daniel
D
Razansky
D000818:Animals; D016923:Cell Death / Q000528:radiation effects*; D045744:Cell Line, Tumor; D004867:Equipment Design; D005453:Fluorescence; D007834:Lasers / Q000009:adverse effects*; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D061848:Optical Imaging; D061088:Photoacoustic Techniques / Q000009:adverse effects*; D038761:Photobleaching; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
Fluorescent protein; Nanosecond laser pulses; Optoacoustic imaging; Photobleaching; Phototoxicity