Multiphoton Microscope

The TriM_Scope is an intravital microscope operating in the multi-photon, confocal, and fluorescent modes. It is highly adaptable, allowing extensive experimental setups to be accomplished on it’s large stage. It is well suited to imaging of live tissues, including tissue cultures and small animals like mice and rats. Advantages of the multi photon mode of imaging include the depth of information, the reduction in photo bleaching and photo toxicity, the clarity of the image, as well as the large adaptable stage.

Multiphoton microscopes differ from the confocal in that the illumination comes from a high power laser operating in the infrared range of photon wavelengths. Instead of a single photon of a higher energy exciting an electron, at least two photons of a longer wavelength, lower energy are required to arrive at the electron simultaneously (or nearly so). Both photons impart energy to the electron, thereby raising it the excited state. In general, it takes two photons of twice the wavelength of the peak excitation wavelength, to excite the fluorescence. Due to spacial and temporal constraints, this doesn’t happen very often, typically 1/1,000,000 as often as the standard fluorescent mechanism occurs, hence the need for a very high powered laser to deliver a large flux of photons to the focal plane of the sample. It is only here that the photons will be compressed enough to have a good probability of arriving at the same time and place to stimulate an electron transition.

The beam path through the multi- photon microscope is similar to the confocal (See link below to Nikon Microscopy U) with the exception it doesn’t use pinholes to eliminate the out of focus fluorescent light. There is no need, since only at the focal plane will the two photons interact with the sample. Detectors are similar, being either photomultiplier or CCDs.

Multiphoton modes have some distinct advantages. Since the infrared photons are of a lower energy, specimen damage is reduced throughout the sample. These photons penetrate the sample to a much greater degree than the visible light photons of the confocal. Furthermore, because few of the infared photons are absorbed except in the minute area of the focal plane, bleaching and toxicity are lessened (see Molecular Expressions). All the fluorescence is coming from this area, there is no light coming from above or below the plane of focus so flare is not a problem. In this way, contrast is markedly increased, resulting in a clearer image than is possible with the confocal. It is worth noting, that resolution is not as good in the multi-photon mode due to the longer wavelength, therefore the larger diffraction limited laser spot.

One of the current applications in the lab include observation and comparison of mouse and rat ovarian cancers using both Optical Coherence Tomography OCT and multi-photon microscopy. The 2 photon microscope has better resolution but with the limitation of about 200 um penetration, The OCT has a lower resolution, but can penetrate farther into the tissue, giving greater detail deep in the tissue.