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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.
Highlights
Second harmonic imaging microscopy (SHIM) involves illuminating non-centrosymmetric structures with an ultrashort pulse of focused laser radiation: the resulting transient power densities can become so high (1018 W m−2) that second-order non-linear frequency doubling occurs
Illumination at 760 nm resulted in the two-photon excitation fluorescence (TPEF) signal dominating the response, shown by the signal detected at longer wavelengths than the second harmonic generation (SHG) signal
This TPEF totally disappeared at longer illumination wavelengths, an SHG signal is still observed with 840 nm (421/10.8 nm) and 940 nm (453/10.8 nm and 474/10.8 nm) illumination
Summary
Second harmonic imaging microscopy (SHIM) involves illuminating non-centrosymmetric structures with an ultrashort pulse of focused laser radiation: the resulting transient power densities can become so high (1018 W m−2) that second-order non-linear frequency doubling occurs. The full permutation symmetry (Kleinman symmetry) is widely assumed in biological SHIM and requires that χ(2) is independent of wavelength[8] Even in this case, wavelength-dependence will arise from factors including the variation in dipole oscillator radiated field amplitude with frequency and 1/λ2 fall in focal spot intensity due to diffraction. This assumption predicts SHG efficiency falls monotonically with wavelength in a material-independent way. Emission from collagen I gels and rat tail tendon sections using excitation wavelengths between 730 nm and 980 nm, describing an identical monotonic fall with wavelength for both materials, this work appears to assess wavelength dependency for forward generated SHG only. Whilst more accurate models have been recently developed[1,19], Boyd’s approach results in a Fourier-transform relation between the axial distribution χ(2)(z) and the 1-D structure factor (Equation 3) which is intuitive and, we believe, captures the essence of our argument: -
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