Multiphoton Microscope

The Intravital microscope is a multi mode microscope, incorporating bright field, fluorescent, confocal and multi-photon capabilities in the same instrument. This allows a flexibility of choice in selecting the best modality for a specific application.

Bright field microscopy is accomplished through the standard microscope setup. An incandescent lamp provides the illumination, which is focused on the sample by the substage condenser. The objective lens magnifies the image, as well as providing the focal point of the microscope. Intermediate lens brings the image to the backplane of the ocular or to the digital camera where the image is recorded.

Fluorescence, as a process, whether occurring under a ultra violet light, in a wide field microscope, a confocal microscope, or a multi-photon microscope all depend on relatively the same process within the sample. (See the Joblonski diagram below)



The exciting short wavelength (higher energy) photons stimulate electrons to a higher energy state, which dwell there for a short time, losing energy over time, then drop back to the ground state, radiating energy of a longer wavelength, (lower energy) ( thereby fluorescence). A given dye or autofluorescent material will have an associated peak excitation and emission wavelength.

Photo-bleaching and photo-toxicity result when some of the photons excite the electrons more than is necessary and beyond the range that they can return to the ground state. The more energy, the greater flux, and the greater area exposed, result in greater damage to the fluorophore and the sample. Some materials are very sensitive to these processes, some are very stable.

Wide field fluorescent microscopy is done through a standard, high pressure mercury bulb epi-illuminating the sample through a filter cube consisting of an excitation filter, a dichroic filter and an emission filter of the appropriate wavelengths. The entire field of the microscope objective, as well as the penetration depth of the beam, is illuminated, the sample is flooded with energetic photons. The detector of the data conveyed by the sample is usually the eye of the observer or digital camera of some design.

Confocal microscopy illuminates the sample with a collimated laser beam, exhibiting appropriate wavelengths to optimally excite the fluorophores present. This might be a helium neon laser, with strong wavelength peaks at 488, 568, and 647 nm, for example. The beam, after passing through a dichroic filter set as in the wide field microscope then passes through a pinhole aperture, and is focused on the sample through the objective. This fine beam is scanned across the sample in a raster pattern, akin to a television screen, by mirrors attached to galvanometers. The photons excite fluorescent light which returns through the objective, the pinhole, the mirrors, back through the dichroic cube, another pinhole, and finally into the detector. The detectors in a confocal may be a PMT or a CCD camera, hooked up to a computer which digitizes the image. Each detector has its advantages and disadvantages depending on the particular application. (Http\\www.) About 95% of the light coming from an image comes from the in-focus plane of the sample and the detector pinhole obliterates that which does not come from this plane. It is this and the pinhole, that allows the optical sectioning, and high resolution which is the confocal’s claim to fame.