Multiphoton Fluorescence Light Microscopy


Multiphoton fluorescence microscopy is a powerful imaging technique that depends on complex quantum mechanical interactions between photons and matter for fluorophore excitation. In conventional fluorescence microscopy, a fluorescent molecule is pumped to an excited state by absorbing a single photon. The molecule subsequently falls back to its ground state by emitting a less energetic photon. This is a linear process of absorbing and emitting energy in the form of single photons. By contrast, multiphoton microscopy is based on nonlinear interactions between light and matter, whereby multiple photons are absorbed to bring single fluorophore molecules to an excited state. Two‐photon fluorescence microscopy is the most commonly used multiphoton imaging technique. In two‐photon microscopy, the fluorescent molecule absorbs two photons simultaneously in a single event, and their combined energies provoke the electronic transition of the molecule to the excited state. Advantages of two‐photon fluorescence, compared to typical single‐photon epifluorescence microscopy, include reduced autofluorescence, deeper tissue penetration, inherent confocality and three‐dimensional (3D) imaging as well as minimised photobleaching and photodamage. Thus, two‐photon microscopy facilitates optical sectioning of thick biological specimens in vivo, which would not be possible with conventional imaging techniques. Recent advances in fluorescence microscopy have expanded the application spectrum and usability of multiphoton imaging, which has become an important and versatile tool in modern biomedical research.

Key Concepts

  • Multiphoton microscopy is based on nonlinear interactions between light and matter.
  • Two‐photon excitation transpires only at the objective lens focal volume, where photon density is high enough to generate sufficient absorption events.
  • Multiphoton microscopy requires the use of ultrafast femtosecond, pulsed laser light sources to achieve appropriate fluorophore excitation conditions at the focal point.
  • Because of sharply focused excitation in two‐photon microscopy, photodamage is reduced and viability of the biological specimen is increased.
  • Multiphoton microscopy uses photons with near infrared wavelengths that are poorly absorbed and less scattered by biological material, allowing deeper light penetration into specimens.
  • As a result of using photons of longer wavelength for excitation, two‐photon fluorescence microscopy is limited to slightly lower resolution, compared with single‐photon confocal microscopy.
  • In two‐photon excitation, photodamage and thermal damage are considerably reduced and become significant only at the focal point.
  • Photoconvertible fluorophores have been developed for multiphoton microscopy applications that allow localised photochemical reactions in a biological sample.
  • Conventional fluorophores exhibit distinct two‐photon absorption spectra, which are not directly related to their single‐photon excitation properties.
  • Two‐photon microscopy is suitable for in vivo imaging and monitoring of neuronal function in freely moving and behaving animals.

Keywords: confocal microscopy; fluorophore; GFP; imaging; infrared light; laser; model organism; multiphoton absorption; nonlinear process; photodamage

Figure 1. Jablonski diagram of one‐photon (a) and two‐photon (b) excitation, which occurs as fluorophores are excited from the ground state to the first electronic states. One‐photon excitation occurs through the absorption of a single photon. Two‐photon excitation occurs through the absorption of two lower‐energy photons via short‐lived intermediate states. After either excitation process, the fluorophore relaxes to the lowest energy level of the first excited electronic states via vibrational processes. The subsequent fluorescence emission process for both relaxation modes is the same.
Figure 2. A schematic representation of the localisation of two‐photon excitation. (a) Infrared light (blue arrow) is focused by an objective lens and fluorescence (red arrow) occurs only at the focal volume. (b) A detailed excitation profile of the two‐photon excitation volume. The full width at half‐maximum of the excitation profile is 0.3 µm along the radial direction (left) and is 0.9 µm along the longitudinal direction (right) at a laser wavelength of 960 nm. (c) A demonstration of the localisation of two‐photon excitation volume. Fluorescein solution is excited by one‐photon excitation (blue arrow) via a 0.1 numerical aperture objective; fluorescence excitation is observed throughout the path of the laser beam. For two‐photon excitation using a second objective with the same numerical aperture (red arrow), fluorescence excitation occurs only from a 3D localised spot.
Figure 3. A schematic drawing of typical components in a two‐photon microscope. This system typically consists of a high‐peak‐power pulsed laser, a high‐throughput scanning microscope and high‐sensitivity detection circuitry.


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Palikaras, Konstantinos, and Tavernarakis, Nektarios(Dec 2015) Multiphoton Fluorescence Light Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002991.pub3]