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.
Spectrally resolved two-photon excited autofluorescence imaging is used to distinguish different cell types and functional areas during dynamic processes in the living gut. Excitation and emission spectra of mucosal tissue and tissue components are correlated to spectra of endogenous chromophores. We show that selective excitation with only two different wavelengths within the tuning range of a Ti:sapphire femtosecond laser system yields excellent discrimination between enterocytes, antigen presenting cells and lysosomes based on the excitation and emission properties of their autofluorescence. The method is employed for time-lapse microscopy over up to 8 h. Changes of the spectral signature with the onset of photodamage are demonstrated, and their origin is discussed.
We present a novel experimental setup to intravitally induce and monitor tissue lesions intravitally at a subcellular level
in murine small intestinal mucosa. Using single 355-nm, 500-ps laser pulses coupled to a two-photon microscope, we
induced optical breakdown with subsequent cavitation bubble formation in the tissue. Imaging was based on spectrally
resolved two-photon excited tissue autofluorescence, while online-dosimetry of the induced microbubbles was done by a
cw probe-beam scattering technique. From the scattering signal, the bubble size and dynamics could be deduced on a ns
time scale. In turn, this signal could be used to control the damage size. This was important for reproducible production
of minute effects in the tissue, despite strong biological variations in tissue response to pulsed laser irradiation.
After producing local UV damage, cells appeared dark, probably due to destruction of mitochondria and loss of
NAD(P)H fluorescence. Within 10 min after cell damage, epithelial cells adjacent to the injured area migrated into the
wound to cover the denuded area, resulting in extrusion of the damaged cells from the epithelial layer. Using the nuclear
acid stain propidium iodide, we could show that UV pulses induced cell membrane damage with subsequent necrosis,
rather than apoptosis. For lesions without disruption of the basement membrane, we did not detect migration of immune
cells toward the injured area within observation periods of up to 5 hours.
This model will be used in further studies to investigate the intrinsic repair system and immune response to laserinduced
lesions of intestinal epithelium in vivo.
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