Combined two-photon excited fluorescence and second-harmonic generation backscattering microscopy of turbid tissues

Aikaterini Zoumi; Alvin T. Yeh; Bruce J. Tromberg

doi:10.1117/12.470701

17 June 2002 Combined two-photon excited fluorescence and second-harmonic generation backscattering microscopy of turbid tissues

Aikaterini Zoumi, Alvin T. Yeh, Bruce J. Tromberg

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Proceedings Volume 4620, Multiphoton Microscopy in the Biomedical Sciences II; (2002) https://doi.org/10.1117/12.470701
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States

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.

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Aikaterini Zoumi, Alvin T. Yeh, and Bruce J. Tromberg "Combined two-photon excited fluorescence and second-harmonic generation backscattering microscopy of turbid tissues", Proc. SPIE 4620, Multiphoton Microscopy in the Biomedical Sciences II, (17 June 2002); https://doi.org/10.1117/12.470701

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