10 July 2003 Effects of femtosecond pulse dispersion precompensation on average power damage thresholds for live cell imaging: implications for relative roles of linear and nonlinear absorption in live cell imaging
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Abstract
Multiphoton microscopy is becoming an increasingly popular modality of laser scanning microscopy for imaging living specimens. In order to improve the signal to noise ratio under the challenging imaging conditions typical of biomedical research, researchers may be forced to resort to measures which substantially increase the amount of energy to which specimens are exposed. Several mechanisms of damage to living cells compete to limit the power window for minimally invasive imaging in multiphoton microscopy; these mechanisms include heating due to linear absorption, phototoxicity related to multiphoton absorption, and optical breakdown due to multiphoton ionization. The relative contribution of each of these factors may change significantly depending on the specimen and imaging parameters. The present study investigates the dominant factors in limiting cell viability at moderate to high-energy fluence levels that may be necessary under non-ideal imaging conditions. The results of this study suggest that heating associated with optical breakdown is an important factor in limiting cell viability under difficult imaging conditions, and that this heating scales in a manner proportional to the energy fluence associated with a given set of scanning parameters. Also significant is the finding that cell viability does not appear to scale proportionally to pulse width in a manner consistent with 2-photon absorption at ultrashort pulse widths and moderate energy fluence levels. These results suggest that the use of a pre-compensation scheme to offset the positive group velocity dispersion due to laser scanning microscope optics is a practical means to increase the signal to noise ratio while minimally impacting cell damage thresholds at ultrafast (sub 200 femtosecond) pulse widths.
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Karl Garsha, "Effects of femtosecond pulse dispersion precompensation on average power damage thresholds for live cell imaging: implications for relative roles of linear and nonlinear absorption in live cell imaging", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.478007; https://doi.org/10.1117/12.478007
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