title: Mitigating thermal mechanical damage potential during two-photon dermal imaging.
abstract: Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.
authors.affiliation: University of Illinois at Urbana-Champaign, Laboratory for Fluorescence Dynamics, Department of Physics, Urbana, Illinois 61801, USA.
authors.forename: Barry R
authors.initials: BR
authors.lastname: Masters
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
authors.forename: Christof
authors.initials: C
authors.lastname: Buehler
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barry
authors.forename: Jason D
authors.initials: JD
authors.lastname: Sutin
authors.forename: William W
authors.initials: WW
authors.lastname: Mantulin
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D002056:Burns / Q000209:etiology* / Q000517:prevention & control*; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D007259:Infrared Rays / Q000009:adverse effects; D007834:Lasers / Q000009:adverse effects; D036641:Microscopy, Fluorescence, Multiphoton / Q000009:adverse effects* / Q000379:methods*; D011829:Radiation Dosage; D018570:Risk Assessment / Q000379:methods; D012307:Risk Factors; D012867:Skin / Q000293:injuries / Q000473:pathology* / Q000528:radiation effects*; D046509:Tissue Culture Techniques