Description

The MFM uses pulsed long-wavelength light to excite fluorophore

Fluorophore

A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...

s within the specimen being observed. The fluorophore absorbs the energy from two long-wavelength photons which must arrive simultaneously in order to excite an electron

Electron

The electron is a subatomic particle with a negative elementary electric charge. It has no known components or substructure; in other words, it is generally thought to be an elementary particle. An electron has a mass that is approximately 1/1836 that of the proton...

 into a higher energy state, from which it can decay, emitting a fluorescence

Fluorescence

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 signal. It differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength, as the summed energies of two long-wavelength exciting photons will produce an emission wavelength shorter than the excitation wavelength.

Multiphoton fluorescence microscopy has similarities to confocal laser scanning microscopy

Confocal laser scanning microscopy

Confocal laser scanning microscopy is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning...

. Both use focused laser beams scanned in a raster pattern to generate images, and both have an optical sectioning

Optical sectioning

Optical sectioning is the process by which a suitably designed microscope can produce clear images of a focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome...

 effect. Unlike confocal microscopes, multiphoton microscopes do not contain pinhole apertures, which give confocal microscopes their optical sectioning quality. The optical sectioning produced by multiphoton microscopes is a result of the point spread function

Point spread function

The point spread function describes the response of an imaging system to a point source or point object. A more general term for the PSF is a system's impulse response, the PSF being the impulse response of a focused optical system. The PSF in many contexts can be thought of as the extended blob...

formed where the pulsed laser beams coincide. The multiphoton point spread function is typically dumbbell-shaped (longer in the x-y plane), compared to the upright rugby-ball shaped point spread function of confocal microscopes.

The longer wavelength, low energy (typically infra-red) excitation lasers of multiphoton microscopes are well-suited to use in imaging live cells as they cause less damage than short-wavelength lasers, so cells may be observed for longer periods with fewer toxic effects. Many researchers are currently working toward better and higher resolution multiphoton imaging developments.

External links

• Molecular Expressions introduction to multiphoton microscopy