Second harmonic generation confocal microscopy of collagen type I from rat tendon cryosections.
We performed second harmonic generation (SHG) imaging of collagen in rat-tendon cryosections, using femtosecond laser scanning confocal microscopy, both in backscattering and transmission geometries. SHG transmission images of collagen fibers were spatially resolved due to a coherent, directional SHG component. This effect was enhanced with the use of an index-matching fluid (n(i) = 1.52). The average SHG intensity oscillated with wavelength in the backscattered geometry (isotropic SHG component), whereas the spectral profile was consistent with quasi-phase-matching conditions in transmission geometry (forward propagating, coherent SHG component) around 440 nm (lambda(p) = 880 nm). Collagen type I from bovine Achilles tendon was imaged for SHG in the backscattered geometry and its first-order effective nonlinear coefficient was determined (|d(eff)| approximately 0.085(+/-0.025)x10(-12)mV(-1)) by comparison to samples of inorganic materials with known effective nonlinear coefficients (LiNbO3 and LiIO3). The SHG spectral response of collagen type I from bovine Achilles tendon matched that of the rat-tendon cryosections in backscattered geometry. Collagen types I, II, and VI powders (nonfibrous) did not show any detectable SHG, indicating a lack of noncentrosymmetric crystalline structure at the molecular level. The various stages of collagen thermal denaturation were investigated in rat-tendon cryosections using SHG and bright-field imaging. Thermal denaturation resulted in the gradual destruction of the SHG signal.
Theodossiou
Theodossis A
TA
Department of Medicine, The Rayne Institute, University College London, London, UK. t.theodossiou@gmail.com
Thrasivoulou
Christopher
C
Ekwobi
Chidi
C
Becker
David L
DL
D000818:Animals; D002417:Cattle; D024042:Collagen Type I / Q000737:chemistry* / Q000648:ultrastructure; D024043:Collagen Type II / Q000737:chemistry / Q000648:ultrastructure; D024142:Collagen Type VI / Q000737:chemistry / Q000648:ultrastructure; D005260:Female; D005629:Frozen Sections; D018613:Microscopy, Confocal; D011489:Protein Denaturation; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013710:Tendons / Q000737:chemistry* / Q000648:ultrastructure
D024042:Collagen Type I; D024043:Collagen Type II; D024142:Collagen Type VI