Biological specimens usually represent a very complex amalgam of tiny structures with differing refractive indices and absorbances. Even if the definition and sensitivity of the microscope are at the maximum, the focal plane is surrounded by these (optically) detrimental structures. Without appropriate measures, we would have a hard time to see anything in ordinary biological samples. Manufacturing thin slices of the sample, usually accomplished by use of microtomes, provides sufficiently clear images, but is not compatible with living objects.
Optical approaches are confocal microscopy and light sheet microscopy. In light sheet microscopy the sample is illuminated by a thin layer orthogonal to the observation, resembling an optical microtome cut. The confocal microscope is based on point-scanning the sample with a diffraction-limited illumination spot and sensing the signal with a diffraction-limited detection spot. The overlay of the two leads to spatial filtering off the extrafocal information.
Another optical option is nonlinear illumination, initially described as two-photon excitation  by M. Göppert-Mayer. Because of the square dependence on light intensity, excitation is constrained to a layer where the photon density is high enough to cause a significant signal: the slice is generated by selective excitation in the focal plane. For fluorescence, all we need to do is to illuminate with wavelengths roughly double the wavelength required for single-photon excitation. As a very beneficial side-effect, scattering is inversely proportional to the fourth power of the wavelength, allowing details deep inside the subject to be imaged with the longer wavelengths .
Illumination for Multiparameter Multiphoton Excitation
A central requirement in biological microscopy is correlation of different signals, for example differently stained structures or metabolic signals. In many cases, two or three fluorescent stains are employed and in some cases even more. Often, different excitation wavelengths are necessary to satisfactorily excite the applied fluorochromes. Although lasers for multiphoton excitation are tunable, simultaneous recording demands a multitude of laser lines injected simultaneously into the beam path. Therefore, it would be desirable to have three or even four independent ports for coupling lasers into the instrument. The TCS SP8 DIVE by Leica Microsystems offers coupling of up to four infrared (