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March 10, 2016

Steve Knight

Tech focus – multi-photon microscopy

Laser Lines’ Steve Knight takes a look at an ever-developing technique, multi photon microscopy

A well established and well used technique, two photon microscopy, or multi-photon fluorescence microscopy, has the ability to create incredibly selective, high-resolution images.

The technique is particularly well-suited to capturing images of live cells as using longer wavelength lasers causes less damage than short-wavelength lasers, enabling cells to be observed for longer periods with fewer toxic effects.

How does it work?

Fluorophores, chemical compounds that fluoresce upon excitation, are introduced into a particular specimen as a dye. A focused laser beam is then used to excite an electron in the fluorophore into a higher energy state, unlike in conventional fluorescence microscopy where the excitation wavelength is longer than the fluorescence wavelength so multiple photons have to be absorbed.

As the electron decays, it emits a fluorescence signal which is captured by the microscope, imaging more deeply and enabling a more precise analysis than single photon microscopy.

The challenge now is to be able to image even more deeply into tissue than is currently possible with two photon microscopy.  This would be of great interest to many researchers, particularly in the area of Neurobiology. Three photon microscopy may well enable this to be achieved.

Conventionally, Ti:sapphire femtosecond lasers have been used for two-photon fluorescence microscopy. However, the maximum wavelength of these IR lasers is 1,080 nm and three photon imaging requires excitation out to ~1,550nm. All is not lost though, as the inclusion of an optical parametric oscillator (OPO) extends the wavelengths up to 1,550 nm making three photon imaging possible.

How can Laser Lines help?

Here at Laser Lines we’re in the enviable position of having the Radiantis Blaze as part of our photonics product portfolio. The Blaze is unique in that it’s the only product on the market to combine an OPO, Ti:Sapphire oscillator and pump laser in one single platform for maximum compactness and usability.

Users are therefore working with a single supplier, single interface and a single piece of equipment, significantly lowering the total cost of ownership of the device and making it an extremely appealing prospect for those in the know.

If you are already using a Ti:Sapphire laser for two photon imaging and also want a hands off, sealed, computer controlled source for three photon imaging, then we still have a solution for you. The Oria IR OPO is compatible with commercial Ti:Sapphire systems and makes it a breeze to extend your excitation capability out to 1,550nm!

For more information on how both the Blaze & Oria could help you, or about any of our Photonics products, contact Steve Knight or his team on 01295 672 588 or

Category: Photonics
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