Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Aug 20;99(17):11014-9.
doi: 10.1073/pnas.172368799. Epub 2002 Aug 12.

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

Aikaterini Zoumi  1 , Alvin Yeh, Bruce J Tromberg
Affiliations

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

Aikaterini Zoumi et al. Proc Natl Acad Sci U S A. .

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.

PubMed Disclaimer

Figures

Fig 1.
Fig 1.
Emission spectra obtained from RAFT collagen for λex = 730, 750, 800, 840, and 880 nm for acquisition time Δt = 60 s. Each spectrum is the average of five measurements acquired at different x–y locations of the sample surface (z = 0 μm). Spectral data obtained for slightly different values of excitation power, P, (namely, 54.2 mW for λex = 730 nm, 57.3 mW for λex = 750 nm, 60.1 mW for λex = 800 nm, 64.1 mW for λex = 840 nm, and 60.3 mW for λex = 880 nm) have been normalized to P = 60.1 mW by using the quadratic dependence of SHG intensity on the excitation power, which was experimentally validated. The spectra have been corrected for the spectral dependence of the microscope objective transmission, the spectrograph grating efficiency, and the transmission of the optical components (filters, dichroic mirrors, etc.) in the optical path of the two-photon system. Inset shows an image obtained from RAFT collagen for λex = 800 nm at z = 0 μm. (Bar = 5 μm.)
Fig 2.
Fig 2.
Color-coded image of immunostained collagen fibers in the RAFT. The image is an overlay of two images obtained from the same sample site by using an SBG39 (322–654 nm) wide-pass emission filter and a 520/40 nm bandpass emission filter for λex = 800 nm, and P = 60 mW. Collagen fibers are shown in cyan, and the spotted staining pattern of the antibody is shown in yellow. (Bar = 3 μm.)
Fig 3.
Fig 3.
Spectral dependence of SHG intensity. The experimental data correspond to the SHG peak intensity values obtained from the corrected spectra in Fig. 1. Each point is the average of five measurements acquired at different x–y locations of the sample surface. Each measurement was performed in triplicate. The solid line corresponds to a nonlinear regression fit of the form ISHG = a + b sin2(2πλ/c + d).
Fig 4.
Fig 4.
SHG images from RAFT collagen at depths of 0, 50, 100, 150, 200, and 230 μm for λex = 800 nm and P = 60 mW (a–f). (Bar = 5 μm.) The corresponding spectra for Δt = 60 s are shown in g. (g Inset) Plot of the log of SHG intensity vs. depth, z (μm).
Fig 5.
Fig 5.
Combined SHG/TPEF images from RAFT collagen only (a), and for collagen and cell (b) for λex = 730 nm, P = 54.2 mW, z = 88 μm. (Bar = 8 μm.) The corresponding emission spectra (Δt = 60 s) are shown in c. SHG image from RAFT collagen only (d), and combined SHG/TPEF image for collagen and cell (e) for λex = 800 nm, P = 60 mW, z = 40 μm in d, and z = 36 μm in e. (Bar = 8 μm.) The corresponding emission spectra (Δt = 60 s) are depicted in f.
Fig 6.
Fig 6.
(Upper) Images of the RAFT (collagen and fibroblast) acquired at λex = 750 nm, for P = 60.2 mW, at the same focal plane z = 80 μm (a–d). Images were obtained from the same sample site by using different filters in front of the PMT: (a) An SBG39 filter (320–620 nm); (b) a 440/40 nm bandpass filter; and (c) a 520/40 nm bandpass filter. Image in d was obtained by overlaying the images in b and c. (Lower) Images from the same site of the RAFT (collagen and fibroblast) acquired at λex = 840 nm, for P = 64.1 mW, at the same focal plane z = 162 μm (e–h). Images e–g were obtained by using the same filters as in a–c. Image in h was obtained by overlaying the images in f and g. (Bar = 6 μm.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Denk W., Strickler, J. H. & Webb, W. W. (1990) Science 248, 73-76. - PubMed
    1. Masters B. R., So, P. T. C. & Gratton, E. (1998) Lasers Med. Sci. 13, 196-203.
    1. Masters B. R. & So, P. T. C. (2001) Opt. Express 8, 2-10. - PubMed
    1. Agarwal A., Coleno, M. L., Wallace, V. P., Wu, W. Y., Sun, C. H., Tromberg, B. J. & George, S. C. (2001) Tissue Eng. 7, 191-202. - PubMed
    1. Diaspro A. & Robello, M. (2000) J. Photochem. Photobiol. B 55, 1-8. - PubMed

Publication types