Two photon or multiphoton laser-scanning microscopy is the latest technique to be introduced commercially in the filed of fluorescence laser scanning microscopy. It takes advantage of an effect discovered by laser developers about 20 years ago.

As applied in the laser-scanning microscope, a diffraction-limited volume (at a focal point) is illuminated with high intensity light at twice the excitation wavelength. The high intensity enables the virtually simultaneous arrival of two photons to raise an electron to an elevated state. The high intensity illumination is attained by focusing a beam from a high energy pulsed laser delivering bursts of 100 femtosecond to 1-2 picosecond pulses at high frequencies (100 MHz). Images are built up by scanning the laser illumination across the specimenand changing the focal plane, as with the conventional confocal laser-scanning microscope. The high intensity illumination necessary for two-photon excitation is only achieved within the focal volume. Thus, there is essentially no fluorescence from outside the focal plane and optical sectioning is obtained intrinsically by the two photon effect, rather than by the use of pinholes in the confocal laser-scanning microscope. The resultant images are free of out-of-focus light and possess higher image contrast because of the non-linear excitation. In addition, photobleaching of indicator and photodamage to the cells is virtually non-existent outside the focal plane, which makes the method particularly attractive for imaging of living cells.

Two photon excitation offers major advantages when working in thick tissue, such as brain slices or developing embryos, due to the dramatically reduced effects of light scattering. This is partly because the longer red and near-IR wavelengths used for two photon illumination penetrate deeper into biological tissue with less absorption and scattering. However, the main advantage comes from the non-linear excitation. The requirement for two coincident (or near coincident) photons to achieve excitation of the fluorophore means that only focused light reaches the required intensities and that scattered light does not cause excitation of the fluorophore. On the return side scattering does not matter, as it does in a confocal scanning microscope, because there is no need to descan the light or use a pinhole in front of the detector. Thus, the design of the two-photon laser scanning microscope makes it inherently insensitive to the effects of light scattering in thick slices, which are normally quite detrimental to the contrast of the final images.

The simultaneous photon “jumping” is not limited to two photons. When three photons arrive simultaneously, the wavelength of the effective excitation light will be one third of that used to create the effect, and so on. Hence the term “multiphoton” has been applied to the technology and there are commercially available systems which will do three photon scanning.

The 2- photon laser scanning system in the Carol Moss Spivak Cell Imaging Facility consists of the following:

A Spectra-Physics Integrated Two-Photon Laser System comprises a Millenia-X-P solid state laser and a Model 3941-M Tsunami Ti:Sapphire laser. The Millenia is a high power, diode pumped solid state laser that can produce >10W continuous power at 532nm. The Tsunami is a regeneratively mode-locked Ti:Sapphire laser and is used in the picosecond configuration. This combination has an exceptionally low amplitude noise and can be tuned between 780 and 900 nm.

It should be noted that the 2-photon effect does not necessarily produce a wavelength input:output ratio of 2:1. The output wavelength varies considerably and the best wavelength for scanning must sometimes be determined empirically.

This laser system is ported to two Leica TCS-SP Confocal Microscopes, one inverted and one fixed-stage upright. Each of these microscopes has argon, krypton, and HeNe lasers providing wavelengths of 488, 568, and 633 nm which cover most of the necessary wavelengths for confocal laser-scanning fluorescence. The fixed stage upright microscope will provide for users who will require either electrophysiological measurements or microinjection for their experiments and should allow them to make recordings from cells near the top of the tissue slices. The microscope is fully equipped for differential interference microscopy used by several users in the infra-red range to visualize their cells, and choose cells for electrical and optical recording. A range of different objectives is included because the needs of the user group range from those who need high N.A. objectives with long working distances to those needing lower power to map molecular and cellular markers over large distances. The inverted microscope will provide for the needs of most other users (normal slide specimens) and especially those who have other live cell or inverted microscope needs. Simultaneous use of the 2-photon laser by both microscopes is possible provided that the users of each microscope setup can use the same infrared beam wavelength. These microscopes were chosen to provide performance and flexibility for a multi-user core facility.

The 2-Photon laser system produces 10.5 W of 532 nm green light from the Millenia laser and 7.5 W of INVISIBLE infrared laser beam radiation and both lasers are Class IV. To use these lasers, you will be required to sign a use consent form and agree to abide by the facility rules for safe use.