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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 2, Iss. 4 — Apr. 1, 2011
  • pp: 805–816

Mechanisms of high-order photobleaching and its relationship to intracellular ablation

S. Kalies, K. Kuetemeyer, and A. Heisterkamp  »View Author Affiliations

Biomedical Optics Express, Vol. 2, Issue 4, pp. 805-816 (2011)

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In two-photon laser-scanning microscopy using femtosecond laser pulses, the dependence of the photobleaching rate on excitation power may have a quadratic, cubic or even biquadratic order. To date, there are still many open questions concerning this so-called high-order photobleaching. We studied the photobleaching kinetics of an intrinsic (enhanced Green Fluorescent Protein (eGFP)) and an extrinsic (Hoechst 33342) fluorophore in a cellular environment in two-photon microscopy. Furthermore, we examined the correlation between bleaching and the formation of reactive oxygen species. We observed bleaching-orders of three and four for eGFP and two and three for Hoechst increasing step-wise at a certain wavelength. An increase of reactive oxygen species correlating with the bleaching over time was recognized. Comparing our results to the mechanisms involved in intracellular ablation with respect to the amount of interacting photons and involved energetic states, we found that a low-density plasma is formed in both cases with a smooth transition in between. Photobleaching, however, is mediated by sequential-absorption and multiphoton-ionization, while ablation is dominated by the latter and cascade-ionization processes.

© 2011 OSA

OCIS Codes
(170.1020) Medical optics and biotechnology : Ablation of tissue
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Laser-Tissue Interactions

Original Manuscript: January 14, 2011
Revised Manuscript: February 18, 2011
Manuscript Accepted: February 20, 2011
Published: March 4, 2011

S. Kalies, K. Kuetemeyer, and A. Heisterkamp, "Mechanisms of high-order photobleaching and its relationship to intracellular ablation," Biomed. Opt. Express 2, 805-816 (2011)

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