Two-photon excitation microscopy

Two-photon excitation microscopy of mouse intestine. Red: actin. Green: cell nuclei. Blue: mucus of goblet cells. Obtained at 780 nm using a Ti-sapphire laser.

Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

Two-photon excitation microscopy typically uses near-infrared (NIR) excitation light which can also excite fluorescent dyes. Using infrared light minimizes scattering in the tissue because infrared light is scattered less in typical biological tissues. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for this technique. Two-photon excitation can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching.[1][2]

Two-photon fluorescence image (green) of a cross section of rhizome colored with lily of the valley. The excitement is at 840 nm, and the red and blue colors represent other channels of multiphoton techniques which have been superimposed.
  1. ^ Denk, Winifried; Strickler, James H.; Webb, Watt W. (6 April 1990). "Two-Photon Laser Scanning Fluorescence Microscopy". Science. 248 (4951): 73–76. Bibcode:1990Sci...248...73D. doi:10.1126/science.2321027. PMID 2321027. S2CID 18431535.
  2. ^ Stockert, Juan Carlos; Blazquez-Castro, Alfonso (2017). "Non-linear Optics". Fluorescence Microscopy in Life Sciences. pp. 642–686. doi:10.2174/9781681085180117010023. ISBN 978-1-68108-518-0.