Two-photon Microscopy (Multiphoton)

Image deep-tissue live cells for extended time periods

Two-photon fluorescence occurs when a fluorophore simultaneously absorbs two photons. Each photon carries approximately half of the energy necessary to excite the fluorophore.

Standard fluorescence microscopy illuminates a sample as far as the excitation light can penetrate. This generates out-of-focus fluorescent light that reduces resolution and obscures fine details. The excitation light may also be absorbed by the sample above and below the focal plane, increasing the risk of photodamage.

Two-photon microscopy eliminates out-of-focus absorption, improving resolution and reducing damage. The penetration depth is also increased as more of the excitation light reaches the desired plane of a sample.

Two-photon excitation happens when a fluorophore simultaneously absorbs two photons. This is achieved using a focussed laser delivering short (“femtosecond”) duration pulses. In this way, a greater photon flux is present at the fluorophore, and the probability that two photons are simultaneously absorbed by the fluorophore increases significantly.

Two-photon microscopes excite fluorophores at the focal plane without absorption elsewhere in the sample. This is achieved using a focussed ultrashort pulsed laser generating a large enough photon density at the focal point for two-photon excitation to occur.

When excitation occurs, the resulting fluorescence is collected by the same objective lens used to illuminate the sample and is reflected off a dichroic mirror onto the detector.

A two-dimensional slice of a sample is obtained by scanning the excitation light across the focal plane (“XY-scanning”). One method for achieving this is by using one or more motorised oscillating mirrors (“galvanometer mirror scanners”). The focal plane can be adjusted by changing the height of the sample relative to the objective lens (“Z-scanning”). Successive slices are computationally layered to generate a three-dimensional image.


As two-photon microscopy eliminates out-of-focus absorption, the technique is successfully used to penetrate deep into living tissue (up to approximately 1 mm). Some example specimens include:

  • brain slices
  • microvasculature
  • embryos
  • retinal tissue
  • tumours
  • osteoarthritis in cartilage
  • fibrous tissue in heart valves

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