A new mode of contrast in biological second harmonic generation microscopy

Abstract

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature.

Passive stiffness of fibrotic skeletal muscle in mdx mice relates to collagen architecture

“…We utilized SHG imaging, which is ideally suited for muscle due to the native signal produced by sarcomeres and collagen (Plotnikov et al 2006a;Chen et al 2012). This also presents a challenge of separating those signals, which we overcame by combining multiple image processing techniques (Liu et al 2013;Green et al 2017;Salick et al 2020). The collagen observed is not consistent with endomysial collagen as there is not a signal surrounding individual muscle fibres (Gillies & Lieber, 2011).…”

X-ray diffraction and second harmonic imaging reveal new insights into structural alterations caused by pressure-overload in murine hearts

“…For instance, one of the TAC hearts presented an active immune response and replacement fibrosis at the left ventricular wall. The regions of high cell infiltration in this TAC heart lacked the acto-myosin diffraction signal, and also the SHG emission from myosin and collagen fibers, which signifies the presence of activated myofibroblasts and the transition of healthy cardiac tissue to a fibrotic subtype 20,21 . By contrast, the two other TAC hearts did not show an active immune response but presented severe hypertrophy in the presence of either interstitial and/or perivascular fibrosis.…”

“…Accordingly, in a coherent optical process the combined absorption of two excitation photons results in the generation of a single photon of exactly twice the energy and half the wavelength of the original excitation wavelength 19 . Interestingly, both collagen and myosin fibers generate intrinsic SHG, making this imaging approach ideal for investigating ECM remodeling and muscle integrity following fibrosis and hypertrophy in cardiac tissue 20,21 . Despite the remarkable advantages of SHG imaging over conventional histology such as high resolution, specificity and reproducibility, the use of this approach for investigating cardiac remodeling in diseased tissue is still in its infancy.…”

Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale

“…Only non-centrosymmetric and fibrillar COL-I structures, in contrast to immature and cytoplasmic COL-I, are able to generate SS-pSHG signal following multiphoton microscopy imaging. 33,34 Strong SS-pSHG signals within nodules as well as thin alternating bright and dark structures were noticed, suggesting a highly ordered molecular arrangement (Figure 2, A). To go further on the collagen organization, observations were made using PLM, allowing the observation down to the microscale of birefringent COL-I.…”