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

TLDR

Multiphoton microscopy provides high‑resolution imaging through nonlinear light–matter interactions, traditionally relying on two‑photon excited fluorescence, while second‑harmonic generation from structural proteins has emerged as a promising contrast mechanism that is limited by transmission‑mode detection, hindering coregistration and in‑vivo thick‑tissue applications. The study demonstrates that backscattering geometry enables easy TPEF/SHG coregistration in unstained tissues across 730–880 nm excitation wavelengths. The combined TPEF/SHG technique was applied to a three‑dimensional organotypic tissue model, with signal origins identified through simultaneous spectroscopy and immunofluorescence confirmation. The authors found that ECM signals are a mix of SHG and TPEF from collagen below 800 nm and purely SHG above 800 nm, that cellular signals match NAD(P)H/FAD spectra, that SHG intensity scales quadratically with power, decays exponentially with depth, and that combining SHG and TPEF yields complementary, noninvasive, spatially localized in‑vivo characterization of cell–ECM interactions.

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 (&lt;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.