Cloning assay thresholds on cells exposed to ultrafast laser pulses

Karsten Koenig; Iris Riemann; Peter Fischer; Thomas P. Becker; Hartmut Oehring; Karl-Juergen Halbhuber

doi:10.1117/12.349214

1 June 1999 Cloning assay thresholds on cells exposed to ultrafast laser pulses

Karsten Koenig, Iris Riemann, Peter Fischer, Thomas P. Becker, Hartmut Oehring, Karl-Juergen Halbhuber

Author Affiliations +

Proceedings Volume 3604, Optical Diagnostics of Living Cells II; (1999) https://doi.org/10.1117/12.349214
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm² and TW/cm². A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P²/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P²/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

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Karsten Koenig, Iris Riemann, Peter Fischer, Thomas P. Becker, Hartmut Oehring, and Karl-Juergen Halbhuber "Cloning assay thresholds on cells exposed to ultrafast laser pulses", Proc. SPIE 3604, Optical Diagnostics of Living Cells II, (1 June 1999); https://doi.org/10.1117/12.349214

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