Abstract

A broad range of excitation wavelengths (730-880nm) was used to demonstrate the co-registration of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) in unstained turbid tissues in reflection geometry. The composite TPEF/SHG microscopic technique was applied to imaging an organotypic tissue model (RAFT). The origin of the image-forming signal from the various RAFT constituents was determined by spectral measurements. It was shown that at shorter excitation wavelengths the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. The cellular signal is due to TPEF at all excitation wavelengths. The reflected SHG intensity followed a quadratic dependence on the excitation power and exhibited a spectral dependence in accordance with previous theoretical studies. Understanding the structural origin of signal provided a stratagem for enhancing contrast between cellular structures, and components of the extracellular matrix. The use of SHG and TPEF in combination provides complementary information that allows non-invasive, spatially localized in vivo characterization of cell-ECM interactions and pathology.

Highlights

  • In this paper we report the combined use of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) for the noninvasive characterization of unstained turbid tissues

  • In order to elucidate the origin of the image-forming signal from two-photon excitation of collagen we have used the RAFT tissue model, which is well characterized

  • Collagen emission spectra acquired from the RAFT surface for λex=730, and 840nm (Fig. 1) exhibit a narrow peak at half the excitation wavelength with a bandwidth (FWHM) that is in accordance to the excitation laser spectral width, and intensity that follows a quadratic dependence on the laser power

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Summary

INTRODUCTION

In this paper we report the combined use of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) for the noninvasive characterization of unstained turbid tissues. Second-harmonic generation (SHG) has been employed as a high-resolution technique for imaging membranes[2,3,4] and endogenous collagen[5,6,7], which is known to exhibit TPEF1;8. This has raised the question of the origin of signal (TPEF or SHG) from biological tissues. We employ spectral measurements in order to elucidate the origin of the image-forming signal from the various tissue constituents, and we use this information to isolate cellular and extracellular matrix signal by spectral filtering

EXPERIMENTAL LAYOUT
TPEF and SHG co-registration from collagen
SHG dependence on excitation wavelength
Separation of collagen from cellular signal
Isolation of cellular and ECM signals by spectral filtering
CONCLUSIONS
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