ABSTRACT
Illumination of the specimen is the most important variable in achieving high-quality images in microscopy. Köhler illumination was rst introduced in 1893 by August Köhler as a method of providing the optimum specimen illumination (Köhler 1893). Köhler illumination has since become the predominant technique for sample illumination in modern scientic light microscopy. It acts to generate an extremely uniform illumination of the sample and ensures that an image of the illumination source (for example, a halogen lamp lament) is not visible in the resulting image, thus allowing the user to realize the microscope’s full potential. With the improvement and accuracy of lens manufacture, some kinds of aberration and blur can be corrected to get a better image quality. However, even if all the optics elements are made perfectly, a resolution limit of about 200 nm still exists in microscopy. This is the so-called diffraction barrier or Abbe diffraction limit (Abbe 1873). Its value is equal to ~0.5λ/ NA, determined only by the wavelength of source light and the numerical aperture (NA) of objective lens. Moreover, the wide-eld geometry nature of light microscopy leads to a certain depth-of-view on the focal plane. When a recording camera is put on the conjugated position of that focal plane, the in-focus and out-of-focus information are fused into a two-dimensional image. The targeted in-focus information cannot be separated from this fusion two-dimensional image directly. That is the reason why the conventional light microscopy cannot obtain optical sectioning, and the captured image is usually low both in contrast and signal-to-noise ratio (SNR).
