title: Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation.
abstract: Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.
authors.affiliation: University of Lübeck, Institute of Anatomy, Ratzeburger Allee 160, 23562, Lübeck, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Klinger
authors.affiliation: University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562, Lübeck, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Krapf
authors.affiliation: University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562, Lübeck, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Orzekowsky-Schroeder
authors.affiliation: University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562, Lübeck, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Koop
authors.affiliation: University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562, Lübeck, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Vogel
authors.affiliation: University of Lübeck, Institute of Biomedical Optics, Peter-Monnik-Weg 4, 23562, Lübeck, GermanycAirway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Germany.
authors.forename: Gereon
authors.initials: G
authors.lastname: Hüttmann
mesh_terms: D000818:Animals; D004751:Enteritis / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005260:Female; D007089:Image Enhancement / Q000295:instrumentation; D007410:Intestinal Diseases; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology* / Q000528:radiation effects; D000069416:Intravital Microscopy / Q000009:adverse effects / Q000295:instrumentation*; D007834:Lasers / Q000009:adverse effects*; D051379:Mice; D008807:Mice, Inbred BALB C; D036641:Microscopy, Fluorescence, Multiphoton / Q000009:adverse effects / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation; D061848:Optical Imaging / Q000009:adverse effects / Q000295:instrumentation*; D011832:Radiation Injuries / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity