Multiphoton (MP) and Second Harmonic Generation (SHG) Imaging

Multiphoton laser scanning with infra-red (IR) lasers supports multicolor fluorescent studies for imaging of living, whole mount or thickly sliced specimens. IR lasers can penetrate more deeply than shorter-wavelength visible lasers and, with no need for pinhole, more light signals are collected, consequently increasing the resolution and contrast of image from deep and thick sample. This provides support for applications where phototoxicity/photobleaching are a concern such as time course studies of living cells and tissues. long working distance objectives and stage allow imaging of large samples, embryos, and animals.

Light by light scattering has been readily observed using the nonlinear response of material systems. Harmonic generation is an instantaneous phenomenon of nonlinear coherent light scattering combining at least two photons and requiring phase-matching conditions. The best-known phenomenon is second harmonic generation (SHG), which is produced only in a non-centrosymmetric medium. SHG occurs without energy absorption. The energy of incident photons is scattered with a harmonic up-conversion. Thus, a laser beam at single frequency ω enters a non-centrosymmetric structure and often some fraction of lights gets converted from single frequency ω to double frequency 2ω. This phenomenon is thus wavelength dependant. Crystals without inversion symmetry are capable of generating a SHG. Notably, filamentous biological proteins with a cylindrical symmetric, e.g. collagen, tubulin or myosin and certain carbohydrate, e.g. starch and cellulose, are also good converters of SHG.

Equipment and Software:

Olympus FV1000 with Prior motorized stage

Spectra-Physics MaiTai IR lasers (690 to 1000 nm) equipped with DeepSee dispersion compensation unit

FV10-ASW

Multiphoton Emission Filter set

  • MRVGR/VR (emission filters; 420-460 nm, 495-540nm & 560-670nm; dichroic 485nm & 570 nm; suitable for BFP/GFP/RFP/DsRed)
  • MRCYR/XR (emission filters; 460-500 nm, 520-560nm & 575-630nm; dichroic 485nm & 570 nm; suitable for CFP/GFP/RFP/DsRed)

 

<Comparison between Confocal and Multi-Photon MS>

Images in upper panel were acquired single photon (559nm) and images in lower panel were acquired with two photon (750nm).  Arabidopsis roots were stained with 10ug/ul Propidium Iodide. Image with Olympus FV1000 by UBC BIF (EunKyoung Lee)

 

 

<SHG images from rat tail collagen fibril>

Imaged with Olympus FV1000 by UBC BIF (EunKyoung Lee)

 

<PIN3-GFP and SHG from starch granules in Arabidopsis columellar root cap>

Starch granules were visualized by SHG of two-photon excitation at 800 nm (green) (left).PIN3-GFP was visualized by two-photon excitation at 900 nm and emission filter 520–560 nm (middle).  Imaged with Olympus FV1000by UBC BIF (EunKyoung Lee)