By recording the light emitted when multiple photons, usually two, interact simultaneously with a fluorescent molecule, multiphoton microscopy can reveal dynamic biological processes occurring deep within biological tissue. This is all because the two excitation photons have half the energy, and thus twice the wavelength, of a single excitation photon. For emitting light at visible wavelengths, this means the excitation photons can be at infrared wavelengths, which are less scattered by biological tissue than visible and ultraviolet wavelengths, and so allow deeper imaging.
With the ability to build up detailed 3D images of biological features at depths of up to 1mm without causing damage, multiphoton microscopy has proved adept at studying both biological samples and living organisms. As such, it is now a critical technique in life science disciplines such as neurophysiology, cell biology, embryology, cancer and immunology.
This Essential Knowledge Briefing provides an introduction to the various forms of multiphoton microscopy and their use in life science. It explains the operation of a typical multiphoton instrument and details the specific practical requirements for different fields of research. It gives examples of how multiphoton microscopy is currently being used by working scientists and previews how it may develop in the future.