When living cells are far below the surface, they pose particularly complex problems for researchers trying to view dynamic life activities in the laboratory. Many fluorescence imaging systems rely on short-wavelength ultraviolet (UV) or blue light, which is then absorbed by the specimen and emitted as visible light. But living tissue scatters so much short-wavelength light that some of the emitted fluorescence from the region of interest does not reach the detector. The deeper the area of interest, the more severe this problem becomes. Indeed, for every specimen, there is a point at which so much scatter occurs that traditional fluorescence imaging techniques are no longer effective. Raising the intensity of the excitation light in order to get more light out of the system can itself be lethal for living systems, as it causes increased photobleaching and phototoxicity. Adding to the problem is the fact that in deep imaging, regions of the specimen above and below the focal plane that are not of interest are exposed to light, causing unwanted fluorescence. Finally, excessive scattering of the excitation light when imaging deep below the specimen's surface results in an image with poor signal-to-noise ratio. These images tend to look soft and dull instead of crisp and full of contrast. Assuming that the scientist's research protocol will not allow the use of thinner-cut sections, deep imaging is still possible via multiphoton microscopy.
Multifocal multiphoton microscopy: A fast and efficient tool for 3-D fluorescence imaging
