Pulse duration and energy dependence of photodamage and lethality induced by femtosecond near infrared laser pulses in Drosophila melanogaster.

The potential application of nonlinear optical imaging diagnosis and treatment using femtosecond laser pulses in humans accentuates the need for studies carried out in whole organisms instead of single cells or cell cultures. While there is a general consensus that in order to minimize the level of photodamage the excitation power has to be kept as low as possible, it has yet to be determined if shorter pulses have greater benefit than longer pulses. Here we evaluate the rate of death in Drosophila melanogaster as the integral parameter related to photodamage resulting from femtosecond near infrared (NIR) laser irradiation under conditions comparable to those used in two-photon excited fluorescence (TPEF) microscopy. We found that the lethality (resulting from photodamage) as a function of laser energy fluence fits a 3-region dose-response curve. The lethality was accompanied with development of necrosis and apoptosis in irradiated tissues. Quantitative analysis showed that the damage has a mostly linear character on energy fluence per pulse, and for a given TPEF signal, shorter (37 fs) pulse duration results in lower lethality than longer (100 fs) pulse duration. These results have important implications for the use of femtosecond NIR laser pulses in microscopy as well as in vivo medical imaging.

[1]  Cynthia A Toth,et al.  A Comparative study of retinal effects from continuous wave and femtosecond mode‐locked lasers , 2002, Lasers in surgery and medicine.

[2]  Yue Zheng,et al.  Damage-Associated Molecular Patterns , and Sterile Inflammation in Cardiovascular Disease , 2011 .

[3]  A. Vogel,et al.  Mechanisms of femtosecond laser nanosurgery of cells and tissues , 2005 .

[4]  J. Haddad.  Redox and oxidant-mediated regulation of apoptosis signaling pathways: immuno-pharmaco-redox conception of oxidative siege versus cell death commitment. , 2004, International immunopharmacology.

[5]  Enrico Gratton,et al.  Mitigating thermal mechanical damage potential during two-photon dermal imaging. , 2004, Journal of biomedical optics.

[6]  Ashleyj . Welch,et al.  Optical-Thermal Response of Laser-Irradiated Tissue , 1995 .

[7]  B. Kaina,et al.  DNA damage-induced cell death by apoptosis. , 2006, Trends in molecular medicine.

[8]  J. Berkson.  Application of the Logistic Function to Bio-Assay , 1944 .

[9]  Avraham Mayevsky,et al.  Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. , 2007 .

[10]  W. Franco,et al.  Hyperthermic injury to adipocyte cells by selective heating of subcutaneous fat with a novel radiofrequency device: Feasibility studies , 2010, Lasers in surgery and medicine.

[11]  Jared P. Stephens,et al.  Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells , 2009, Nucleic acids research.

[12]  Marcos Dantus,et al.  Photobleaching and photoenhancement of endogenous fluorescence observed in two-photon microscopy with broadband laser sources , 2010 .

[13]  W. Webb,et al.  Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  K. König,et al.  Multiphoton microscopy in life sciences , 2000, Journal of microscopy.

[15]  S W Hell,et al.  Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. , 1999, Biophysical journal.

[16]  P. So,et al.  Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. , 1997, Optics letters.

[17]  C. J. Fecko,et al.  Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser. , 2011, Journal of photochemistry and photobiology. B, Biology.

[18]  N. Geacintov,et al.  Multiphoton Near‐Infrared Femtosecond Laser Pulse‐Induced DNA Damage With and Without the Photosensitizer Proflavine , 1999, Photochemistry and photobiology.

[19]  J. Abrams,et al.  Guardian ancestry: fly p53 and damage-inducible apoptosis , 2000, Cell Death and Differentiation.

[20]  E. Neher,et al.  Highly nonlinear photodamage in two-photon fluorescence microscopy. , 2001, Biophysical journal.

[21]  R. Meldrum,et al.  Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three‐photon near‐infrared absorption , 2003, EMBO reports.

[22]  O. Trentz,et al.  Roles of histamine, complement and xanthine oxidase in thermal injury of skin. , 1989, The American journal of pathology.

[23]  Timothy A. Skimina,et al.  Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Maria Smedh,et al.  Multiphoton laser scanning microscopy on non-melanoma skin cancer: morphologic features for future non-invasive diagnostics. , 2008, The Journal of investigative dermatology.

[25]  T. Buttke,et al.  Oxidative stress as a mediator of apoptosis. , 1994, Immunology today.

[26]  Watt W Webb,et al.  Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. , 2002, Biophysical journal.

[27]  K. König,et al.  Cell damage by near-IR microbeams , 1995, Nature.

[28]  K J Halbhuber,et al.  Femtosecond near-infrared laser pulses elicit generation of reactive oxygen species in mammalian cells leading to apoptosis-like death. , 2001, Experimental cell research.

[29]  C. J. Fecko,et al.  DNA multiphoton absorption generates localized damage for studying repair dynamics in live cells. , 2011, Biophysical journal.

[30]  G. Gravante,et al.  Inverse relationship between the apoptotic rate and the time elapsed from thermal injuries in deep partial thickness burns. , 2008, Burns : journal of the International Society for Burn Injuries.

[31]  Jyj Wang,et al.  DNA damage and apoptosis , 2001, Cell Death and Differentiation.

[32]  M. Gu,et al.  Three-dimensional autofluorescence spectroscopy of rat skeletal muscle tissue under two-photon excitation. , 1999, Applied optics.

[33]  C. Kallenberg,et al.  Apoptosis in Human Skin: Role in Pathogenesis of Various Diseases and Relevance for Therapy , 2005, Annals of the New York Academy of Sciences.

[34]  Stefan Puschmann,et al.  Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm. , 2008, Journal of biomedical optics.

[35]  Curtis F. Chapman,et al.  Evidence for localized cell heating induced by infrared optical tweezers. , 1995, Biophysical journal.

[36]  P O'Neill,et al.  Use of near infrared femtosecond lasers as sub-micron radiation microbeam for cell DNA damage and repair studies. , 2010, Mutation research.

[37]  D. Meyerholz,et al.  Apoptosis is differentially regulated by burn severity and dermal location. , 2010, The Journal of surgical research.

[38]  S. Botchway,et al.  Induction of Persistent Double Strand Breaks Following Multiphoton Irradiation of Cycling and G1‐arrested Mammalian Cells—Replication‐induced Double Strand Breaks , 2008, Photochemistry and photobiology.

[39]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[40]  G. Arturson,et al.  ACUTE CHANGES IN CAPILLARY FILTRATION AND DIFFUSION IN EXPERIMENTAL BURN INJURY. , 1964, Acta physiologica Scandinavica.

[41]  Jeroen A. A. Demmers,et al.  Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4 , 2006, Proceedings of the National Academy of Sciences.

[42]  Warren R. Zipfel,et al.  Feasibility of using multiphoton excited tissue autofluorescence for in vivo human histopathology. , 2010, Biomedical optics express.

[43]  K J Halbhuber,et al.  Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes. , 1999, Optics letters.

[44]  O. Trentz,et al.  Role of xanthine oxidase in thermal injury of skin. , 1989, The American journal of pathology.

[45]  B R Masters,et al.  Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. , 1997, Biophysical journal.