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

@article{citeulike:2224124, abstract = {10.1073/pnas.172368799 Multiphoton microscopy relies on nonlinear light\^{a}€“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 \^{a}€œintravital\^{a}€ 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 thick-tissue applications. In this study, we use a broad range of excitation wavelengths (730\^{a}€“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 characterization of cell\^{a}€“ECM interactions in unstained thick tissues.}, author = {Zoumi, Aikaterini and Yeh, Alvin and Tromberg, Bruce J.}, citeulike-article-id = {2224124}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.172368799}, citeulike-linkout-1 = {http://www.pnas.org/content/99/17/11014.abstract}, citeulike-linkout-2 = {http://www.pnas.org/content/99/17/11014.full.pdf}, citeulike-linkout-3 = {http://www.pnas.org/cgi/content/abstract/99/17/11014}, citeulike-linkout-4 = {http://view.ncbi.nlm.nih.gov/pubmed/12177437}, citeulike-linkout-5 = {http://www.hubmed.org/display.cgi?uids=12177437}, day = {20}, doi = {10.1073/pnas.172368799}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {multiphoton\_microscopy}, month = {August}, number = {17}, pages = {11014--11019}, posted-at = {2009-11-09 13:20:47}, priority = {2}, title = {Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence}, url = {http://dx.doi.org/10.1073/pnas.172368799}, volume = {99}, year = {2002} }

TY - JOUR ID - citeulike:2224124 L3 - citeulike-article-id:2224124 N2 - 10.1073/pnas.172368799 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 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 characterization of cell–ECM interactions in unstained thick tissues. IS - 17 JF - Proceedings of the National Academy of Sciences of the United States of America EP - 11019 TI - Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence VL - 99 SP - 11014 KW - multiphoton_microscopy AU - Zoumi, Aikaterini AU - Yeh, Alvin AU - Tromberg, Bruce PY - 2002/08/20/ UR - http://dx.doi.org/10.1073/pnas.172368799 ER -