Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.
Journal: 2002/September - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Multiphoton microscopy relies on nonlinear light-matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue "intravital" imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730-880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), 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. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell-ECM interactions in unstained thick tissues.
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Proc Natl Acad Sci U S A 99(17): 11014-11019

Imaging cells and extracellular matrix <em>in vivo </em>by using second-harmonic generation and two-photon excited fluorescence

Laser Microbeam and Medical Program (LAMMP), Beckman Laser Institute, and Center for Biomedical Engineering, University of California, Irvine, CA 92612
To whom reprint requests should be addressed. E-mail: ude.icu.ilb@grebmort.
Communicated by Peter M. Rentzepis, University of California, Irvine, CA
Communicated by Peter M. Rentzepis, University of California, Irvine, CA
Received 2002 Jan 20; Accepted 2002 Jun 20.

Abstract

Multiphoton microscopy relies on nonlinear light–matter interactions to provide contrast and optical sectioning capability for high-resolution imaging. Most multiphoton microscopy studies in biological systems have relied on two-photon excited fluorescence (TPEF) to produce images. With increasing applications of multiphoton microscopy to thick-tissue “intravital” imaging, second-harmonic generation (SHG) from structural proteins has emerged as a potentially important new contrast mechanism. However, SHG is typically detected in transmission mode, thus limiting TPEF/SHG coregistration and its practical utility for in vivo thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730–880 nm) to demonstrate that TPEF/SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF/SHG technique was applied to imaging a three-dimensional organotypic tissue model (RAFT). The structural and molecular origin of the image-forming signal from the various tissue constituents was determined by simultaneous spectroscopic measurements and confirming immunofluorescence staining. Our results show that at shorter excitation wavelengths (<800 nm), 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. Endogenous cellular signals are consistent with TPEF spectra of cofactors NAD(P)H and FAD at all excitation wavelengths. The reflected SHG intensity follows a quadratic dependence on the excitation power, decays exponentially with depth, and exhibits a spectral dependence in accordance with previous theoretical studies. The use of SHG and TPEF in combination provides complementary information that allows noninvasive, spatially localized in vivo characterization of cell–ECM interactions in unstained thick tissues.

Abstract

Since its introduction by Denk et al. (1), two-photon excited fluorescence (TPEF) has been widely used for imaging structure and dynamic interactions in biological tissues (2–5). Although second-harmonic generation (SHG) in biological tissues was first demonstrated two decades ago (6–8), SHG has only recently been used for biological imaging applications (9–12). Because TPEF and SHG involve different contrast mechanisms, they can be used in tandem to provide complementary information regarding tissue structure and function. Specifically, SHG signals depend on the orientation, polarization, and local symmetry properties of chiral molecules, whereas TPEF results from the nonlinear excitation of molecular fluorescence.

The primary tissue constituent responsible for SHG is collagen (9, 13, 14), which is also a well documented source of tissue autofluorescence (4, 15). This fact has created uncertainty over whether the image-forming signal from two-photon excitation of collagen in biological tissues is TPEF or SHG and prompts further investigation into the precise origin of signal in the nonlinear microscopy of tissues. Detailed characterization of the collagen signal is particularly important because collagen is the most abundant structural protein in higher vertebrates, and structural modifications of the fibrillar matrix are associated with various physiologic processes such as aging, diabetes, wound healing, and cancer (16–18).

Most studies using nonlinear microscopic techniques for biological imaging have relied on TPEF. Recently, a combination of TPEF and SHG has been implemented for the study of cells stained with exogenous probes possessing large molecular anisotropy and second-order nonlinearity (10, 13, 19–21). Combined TPEF/SHG has been demonstrated for thin tissue sections (22), but not for the more practical case of thick, unstained living specimens because, although TPEF is measured in epi-illumination geometry, the forward propagating nature of the phase-coherent SHG signal seems to restrict SHG microscopy to a transmission mode of detection. In fact, under certain conditions, a significant amount of backscattering second-harmonic light can be generated at the interfaces between nonlinear and linear media (23–27). Reflected SHG signals provided the basis for the development of the first in vivo SHG tissue tomography (28, 29). The results obtained from this approach, however, were characterized by relatively poor resolution, lack of structural detail, and prohibitively long acquisition times (0.5–2.8 h).

In this study, we have modified a conventional TPEF laser-scanning microscope to observe the complete spectral content of multiphoton microscopy signals. Images and spectral measurements from two-photon excitation of an unstained organotypic tissue model (RAFT) are used to demonstrate the coregistration of SHG and TPEF from collagen across a broad excitation wavelength range in reflection geometry. The determination of the origin of signal from both cellular and ECM components is accomplished and shown to provide excellent contrast-enhancing opportunities for thick-tissue intravital imaging. Furthermore, the dependence of the detected SHG intensity on excitation wavelength and imaging depth is compared with a theoretical model and proposed as a means for extracting quantitative structural information from the tissue.

Acknowledgments

We thank T. B. Krasieva and C.-H. Sun for helpful discussions and technical assistance. This work was supported by the National Institutes of Health Laser Microbeam and Medical Program (P41RR-01192), the Department of Energy (DOE DE-FG03–91ER61227), Air Force Office of Scientific Research Medical Free Electron Laser Program (F49620–00-1–0371), and National Institutes of Health, Carcinogenesis Training Grant CA-09054 (to A.Y.).

Acknowledgments

Abbreviations

  • SHG, second-harmonic generation

  • TPEF, two-photon excited fluorescence

  • λex, excitation wavelength

  • ECM, extracellular matrix

  • z, depth into the sample

  • PMT, photomultiplier tube

Abbreviations

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