Detection of collagen fibers using Second harmonic generation (SHG) imaging

One remarkable ability of multiphoton microscopy is to provide images with micrometer 3 -dimensional (3D) resolution from within intact biological tissues. An emerging application is the observation of unstained live tissue, based on endogenous sources of nonlinear signals. Imaging intrinsic signals with molecular or structural specificity can be achieved by combining twophoton-excited fluorescence (2PEF) microscopy, second harmonic generation (SHG) microscopy, and spectrally selective detection. Endogenous sources of signal for tissue 2PEF imaging are the same as those excited by conventional one-photon absorption with near-UV or blue excitation: NAD(P)H, flavoproteins, elastin fibers, etc.However, two-photon excitation provides superior imaging depth compared with confocal/conventional microscopy[1].

In a recent study, we tried to image arterial collagen found in the media using SHG. However, it seems that there is a technical problem. Most of collagen found in the media layer of arterial wall is consisted of type IV collagen which is not detectable with SHG.however, the fibrillar collagen in adventitia (mostly type I and III) is detectable. (looking at the figure shown from ref. 1 ,in green is the collagen in adventitia and in red one find the elastin laminas in media of the vessel wall.) Here is my question: Has anyone of you ever tried to image the collagen in media in an unstained tissue using SHG?

[1] T. Boulesteix et al. , " Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure" Cytometry Part A, 69A(1), pp. 20-26.

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