Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation.

Antje Klinger,Norbert Koop,Lisa Krapf,Alfred Vogel,Regina Orzekowsky-Schroeder,Gereon Hüttmann

doi:10.1117/1.jbo.20.11.116001

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.

Highlights

Nonlinear microscopy has opened a new door to intravital imaging
Autofluorescence 2-photon (A2P) microscopy offers the ability for high-resolution subcellular imaging based only on intrinsic molecular signals in cells, such as NAD(P)H [reduced nicotinamide adenine dinucleotide(phosphate) and flavin adenine dinucleotide (FAD)], and reveals most, if not all, of the cells and tissues of the small intestinal mucosa.[3]
As there are no additional fluorescent markers applied to the tissue, A2P microscopy may in the future be used for clinical diagnosis of tumors or other skin diseases.[4,5]

Summary

Introduction

Nonlinear microscopy has opened a new door to intravital imaging. Selective excitation of fluorescence by 2-photon absorption visualizes biological processes in animals and humans in real time as they occur in their native environment.[1]. The laser wavelength has to be changed between 720 and 850 nm for optimal excitation of naturally occurring fluorophores, such as NAD(P)H, flavines, lipopigments, and second-harmonic generation (SHG) of collagen.[6] If additional fluorescent proteins or exogenous dyes are employed, an even larger tuning range may be useful.[7,8] For optimal imaging of multiple fluorophores, the specimen has to be scanned several times with different excitation wavelengths at the expense of reduced imaging speed and increased motion artifacts. With a 100 nm bandwidth around a center wavelength of 780 nm and a pulse width below 13 fs, the autofluorescence of skin has been imaged ex vivo.[11] in that study, the excitation flux was not decreased.

Methods

Results

Conclusion