A new mode of contrast in biological second harmonic generation microscopy

作者：Green Nicola H^*; Delaine Smith Robin M; Askew Hannah J; Byers Robert; Reilly Gwendolen C; Matcher Stephen J

来源：Scientific Reports, 2017, 7(1): 13331.

DOI：10.1038/s41598-017-13752-y

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.

• 出版日期2017-10-17