Ultrastructure and reproduction behaviour of single CHO‐K1 cells exposed to near infrared femtosecond laser pulses

Abstract

Summary:In the present work, the authors investigated ultrastructural changes as well as the reproduction behaviour of preselected single CHO-K1 cells exposed to 170 femtosecond laser pulses at different power output levels in comparison with cells outside the illumination volume. The ultrashort laser pulses were provided by an 80 MHz Ti:sapphire laser at 780 nm. The cells were scanned ten times with a scan rate of 1/16 s -1 . Single CHO-K1 cells exposed to low mean power of 2 mW revealed no significant changes in ultrastructure after laser exposure. In some cases, changes of mitochondria with slight disordering of cristae were found. Cytoplasm was filled with vesicles that seemed to be released from Golgi stacks. Cells irradiated with higher powers demonstrated more dramatic changes in ultrastructure. A considerable number of swollen mitochondria in conjunction with loss of cristae was observed. The main event of mitochondrial changes was the formation of electron dense bodies in the mitochondrial matrix. In addition, lumen of endoplasmatic reticulum was enlarged. Highest applied mean laser power of 12.5 mW lead to complete destruction of mitochondria and their transformation to electron dense structures containing membrane material. Compared with cell targets irradiated with 2 mW mean power, the release of vesicles from Golgi stacks seemed to be rather moderate. Cells localised outside the laser beam revealed no ultrastructural changes. Low mean laser power at 2 mW was unable to impair the reproduction behaviour of CHO-K1 cells. At higher laser power output levels, CHO-K1 cells started to delay cell division. At 12.5 mW, no cell division occurred. The obtained results may be helpful in recommending parameters for safe femtosecond laser microscopy of living specimens.

Imaging of brain and brain tumor specimens by time-resolved multiphoton excitation microscopy ex vivo1

“…In contrast to conventional one-photon laser scanning microscopy, femtosecond pulsed laser microscopy of living specimens can be performed at peak intensities of 200 GW/cm 2 with no sign of structural or functional photodamage. This has been demonstrated for cells in monolayer culture as well as for mammalian embryos and human skin (Masters et al, 1997(Masters et al, , 1998Oehring et al, 2000;Squirrell et al, 1999;Tyrell and Keyse, 1990). Therefore, intraoperative in vivo multiphoton microscopy of brain tissue conceivably could provide a high-resolution, noninvasive diagnostic tool.…”

Advances and challenges in label-free nonlinear optical imaging using two-photon excitation fluorescence and second harmonic generation for cancer research

“…Due to high peak power albeit for pulse duration of femto-seconds, NLOI can induce generation of damaging reactive oxygen species and localized heat effects, leading to loss of cell viability and cell death [43,[53][54][55][56][57]. These thermo-chemical effects are however confined spatially just to the imaged zones, making it no less harmful than an invasive tissue biopsy.…”

Estimating the risk of squamous cell cancer induction in skin following nonlinear optical imaging

“…These lasers can supply high irradiance in the range of GW cm À2 for transient durations as short as 10 À9 to 10 À15 seconds within the focal volume. Even so, during an ultrashort pulse duration, high peak irradiance delivered may still damage the irradiated cells by eliciting a variety of undesired biological responses [4][5][6][7][8][9][10]. However, the mentioned harmful cellular effects are restricted to the irradiated tissue and its immediate surroundings and do not have long term effects.…”