Introduction to Microscopy
Microscopes are specialized optical instruments designed to produce magnified images of specimens that are too small to be seen with the naked eye. In addition to complex designs featuring objectives and condensers, microscopes also consists of very simple single-lens instruments that are often hand-held, such as a common magnifying glass.
How the Microscope Forms Images
Optical microscopes belong to a class of instruments that are said to be diffraction limited, meaning that resolution is determined in part by the number of diffraction orders created by the specimen that can be successfully captured by the objective and imaged by the optical system.
Numerical Aperture and Resolution
The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. Resolution is determined by the number of diffracted wavefront orders captured by the objective.
The Point Spread Function
The ideal point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane of a microscope (or other diffraction-limited optical instrument) through a high numerical aperture (NA) objective or lens system.
Illumination and the Microscope Optical Train
The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. Illumination of the specimen is the most important controllable variable in achieving high-quality images in microscopy and digital imaging.
Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Köhler illumination was first introduced in 1893 by August Köhler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.