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Long‐term monitoring of live cell proliferation in presence of PVP‐Hypericin: a new strategy using ms pulses of LED and the fluorescent dye CFSE
Author(s) -
PENJWEINI ROZHIN,
LOEW HANS G.,
HAMBLIN MICHAEL R.,
KRATKY KARL W.
Publication year - 2012
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2011.03555.x
Subject(s) - photosensitizer , fluorescence , photobleaching , live cell imaging , hypericin , fluorescence recovery after photobleaching , fluorescence lifetime imaging microscopy , chemistry , biophysics , cell , photochemistry , optics , biology , biochemistry , physics , pharmacology
Summary During fluorescent live cell imaging it is critical to keep excitation light dose as low as possible, especially in the presence of photosensitizer drugs, which generate free radicals upon photobleaching. During fluorescent imaging, stress by excitation and free radicals induces serious cell damages that may arrest the cell cycle. This limits the usefulness of the technique for drug discovery, when prolonged live cell imaging is necessary. This paper presents a strategy to provide gentle experimental conditions for dynamic monitoring of the proliferation of human lung epithelial carcinoma cells (A549) in the presence of the photosensitizer Polyvinylpyrrolidone‐Hypericin. The distinctive strategy of this paper is based on the stringent environmental control and optimizing the excitation light dose by (i) using a low‐power pulsed blue light‐emitting diode with short pulse duration of 1.29 ms and (ii) adding a nontoxic fluorescent dye called carboxyfluorescein‐diacetate‐succinimidyl‐ester (CFSE) to improve the fluorescence signals. To demonstrate the usefulness of the strategy, fluorescence signals and proliferation of dual‐marked cells, during 5‐h fluorescence imaging under pulsed excitation, were compared with those kept under continuous excitation and nonmarked reference cells. The results demonstrated 3% cell division and 2% apoptosis due to pulsed excitation compared to no division and 85% apoptosis under the continuous irradiation. Therefore, our strategy allows live cell imaging to be performed over longer time scales than with conventional continuous excitation.