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The observation of sub-wavelength structures with microscopes is difficult because of the Abbe diffraction limit.Ernst Abbe found in 1873, [2] and expressed as a formula in 1882, [3] that light with wavelength , traveling in a medium with refractive index and converging to a spot with half-angle will have a minimum resolvable distance of
Confocal microscopes work on the principle of point excitation in the specimen (diffraction limited spot) and point detection of the resulting fluorescent signal. A pinhole at the detector provides a physical barrier that blocks out-of-focus fluorescence. Only the in-focus, or central spot of the Airy disk, is recorded.
There is a diffraction-limited resolution depending on incident wavelength; in visible range, the resolution of optical microscopy is limited to approximately 0.2 micrometres (see: microscope) and the practical magnification limit to ~1500x.
The diffraction limit is a feature of conventional lenses and microscopes that limits the fineness of their resolution depending on the illumination wavelength and the numerical aperture (NA) of the objective lens. Many lens designs have been proposed that go beyond the diffraction limit in some way, but constraints and obstacles face each of them.
All optical microscopes are diffraction-limited because of the wave nature of light. Current research focuses on techniques to go beyond this limit known as the Rayleigh criterion. The use of SIL can achieve spatial resolution better than the diffraction limit in air, for both far-field imaging [3] [4] and near-field imaging.
STED microscopy is one of several types of super resolution microscopy techniques that have recently been developed to bypass the diffraction limit of light microscopy to increase resolution. STED is a deterministic functional technique that exploits the non-linear response of fluorophores commonly used to label biological samples in order to ...
The limit of optical resolution in a conventional microscope, the so-called diffraction limit, is in the order of half the wavelength of the light used to image.Thus, when imaging at visible wavelengths, the smallest resolvable features are several hundred nanometers in size (although point-like sources, such as quantum dots, can be resolved quite readily).
Diffraction can also be a concern in some technical applications; it sets a fundamental limit to the resolution of a camera, telescope, or microscope. Other examples of diffraction are considered below.