Second-harmonic imaging microscopy

Second-harmonic imaging microscopy (SHIM) is based on a nonlinear optical effect known as second-harmonic generation (SHG). SHIM has been established as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function.[1] A second-harmonic microscope obtains contrasts from variations in a specimen's ability to generate second-harmonic light from the incident light while a conventional optical microscope obtains its contrast by detecting variations in optical density, path length, or refractive index of the specimen. SHG requires intense laser light passing through a material with a noncentrosymmetric molecular structure, either inherent or induced externally, for example by an electric field.[2]

Second-harmonic light emerging from an SHG material is exactly half the wavelength (frequency doubled) of the light entering the material. While two-photon-excited fluorescence (TPEF) is also a two photon process, TPEF loses some energy during the relaxation of the excited state, while SHG is energy conserving. Typically, an inorganic crystal is used to produce SHG light such as lithium niobate (LiNbO3), potassium titanyl phosphate (KTP = KTiOPO4), and lithium triborate (LBO = LiB3O5). Though SHG requires a material to have specific molecular orientation in order for the incident light to be frequency doubled, some biological materials can be highly polarizable, and assemble into fairly ordered, large noncentrosymmetric structures. While some biological materials such as collagen, microtubules, and muscle myosin[3] can produce SHG signals, even water can become ordered and produce second-harmonic signal under certain conditions, which allows SH microscopy to image surface potentials without any labeling molecules.[2] The SHG pattern is mainly determined by the phase matching condition. A common setup for an SHG imaging system will have a laser scanning microscope with a titanium sapphire mode-locked laser as the excitation source. The SHG signal is propagated in the forward direction. However, some experiments have shown that objects on the order of about a tenth of the wavelength of the SHG produced signal will produce nearly equal forward and backward signals.

Second-harmonic image of collagen (shown in white) in liver
  1. ^ Juan Carlos Stockert, Alfonso Blázquez-Castro (2017). "Chapter 19 Non-Linear Optics". Fluorescence Microscopy in Life Sciences. Bentham Science Publishers. pp. 642–686. ISBN 978-1-68108-519-7. Retrieved 24 December 2017.
  2. ^ a b Roesel, D.; Eremchev, M.; Schönfeldová, T.; Lee, S.; Roke, S. (2022-04-18). "Water as a contrast agent to quantify surface chemistry and physics using second harmonic scattering and imaging: A perspective". Applied Physics Letters. 120 (16). AIP Publishing: 160501. Bibcode:2022ApPhL.120p0501R. doi:10.1063/5.0085807. ISSN 0003-6951. S2CID 248252664.
  3. ^ Nucciotti, V.; Stringari, C.; Sacconi, L.; Vanzi, F.; Fusi, L.; Linari, M.; Piazzesi, G.; Lombardi, V.; Pavone, F. S. (2010). "Probing myosin structural conformation in vivo by second-harmonic generation microscopy". Proceedings of the National Academy of Sciences. 107 (17): 7763–7768. Bibcode:2010PNAS..107.7763N. doi:10.1073/pnas.0914782107. ISSN 0027-8424. PMC 2867856. PMID 20385845.