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

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 TPEF͞SHG 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 TPEF͞SHG coregistration can easily be achieved in unstained tissues by using a simple backscattering geometry. The combined TPEF͞SHG technique was applied to imaging a threedimensional 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. S ince 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...

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