Multiphoton Microscopy and Second Harmonic Generation

Endogenously fluorescent image on Ultima courtesy of Joseph Szulczsewski, and David InmanKeely Lab and Laboratory for Optical and Computational Instumentation at the University of Wisconsin-Madison shows mouse tumor model with endogenously fluorescent NADH (blue), FAD (red) and collagen (green) by second harmonic generation.

All multiphoton excitation microscopy systems at LOCI are designed to collect complimentary second harmonic generation data, including the Spectral Lifetime Multiphoton Microscope (SLIM), the Optical Workstation (OWS), Optical Workstation II (OWS II), LINKUltima, and the Compact Automated Multiphoton Microscope (CAMM), and Ultra.

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Article by Kristy Wendt supplemented with videos by Aprameya Mysore

Multiphoton (or two-photon) excitation microscopy permits deeper optical sectioning and selective fluorophore excitation of whole organisms, intact tissue and living cells.  Two-photon excitation occurs when two lower-energy photons are simultaneously absorbed in a single, quantized event. Only the most photon-dense focal plane in the sample fluoresces, so light scattering and out-of-focus light are reduced compared to single-photon techniques, such as laser scanning confocal microscopy. 

This is achieved with a mode-locked infrared laser focused through an objective lens (see video above) that concentrates photons spatially in a select laser focal plane of the tissue so that excitation is confined to the optical section being observed, and temporally with individual pulses of the laser so the average laser power required to generate simultaneous photon absorption is lower, reducing phototoxicity and photobleaching compared to single-photon techniques, like laser scanning confocal microscopy. Video above by Aprameya Mysore is seen most clearly via YouTube. 


Second harmonic generation (SHG) microscopy uses same-frequency photons that interact with a noncentrosymmetric material and are combined to form new photons with twice the energy. In biological tissue, type I and II fibrillar collagen, microtubule assemblies -- including centrosomes and mitotic spindles -- and muscle myosin all endogenously produce a SHG signal without exogenous probes.  Efficiency of the photon frequency doubling effect is interpreted as contrast in imaging and SHG can be used independently or in conjunction with other imaging techniques -- such as multiphoton microscopy -- to show cell and tissue structure and function.  Video above by Aprameya Mysore is seen most clearly via YouTube.      

SHG microscopes at LOCI use circular polarization to optimize signal intensity, which in collagen is dependent on the angle between polarization of light and fiber direction, ranging from zero for zero angle and maximum for ninety degrees. Circular polarization potentially stimulates multidirectional collagen fibers with the same beam power to enhance visibility and contrast.  Video above by Aprameya Mysore is most clearly seen via YouTube.