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Resolution | Resolution

Key Words: confocal, N.A., signal-to-noise-ratio, fluorescence, deconvolution, depth-of-field, objectives, condenser, oil-immersion objectives, water-immersion objectives

Definition:Resolution can be defined as the smallest distance between two points at which they appear as two distinct points rather than as a single blur

TECHNOLOGY:

Overall, the resolving power of a light microscope is in the region of about 0.2 microns although smaller features can be identified with the help of fluorescent probes. Resolution in microscopy is dependent on the quality of the microscope's optical system and the wavelength of light used to construct the image - shorter wavelengths provide better resolution.

Numerical aperture (N.A.) determines the resolving power of an objective, but the total resolution of the microscope system depends also on other components such as the N.A. of the condenser, the correct alignment of all components in the system, and the imaging medium (see oil-immersion and water-immersion objectives). In general, resolution increases with magnification but the combination of high N.A. and high magnification can result in a narrow depth of field and short working distance.

Microscope resolution is related to contrast and image 'sharpness', which in turn, are limited by signal-to-noise ratio. A number of mathematical algorithms in the form of image processing 'deconvolution' software may improve perceived sharpness and image interpretation.

APPLICATIONS:

Resolution determines what the microscope user is able to distinguish and is, therefore, of paramount importance in imaging applications: the greater the resolution of the system, the more information can be determined from the image. The level of resolution required will depend on the imaging application; however, most microscope systems are used successfully below their full resolving capabilities. For the most demanding fluorescence applications, for example using low light and / or thicker specimens, confocal imaging can greatly improve resolution (compared with widefield techniques) by blocking the out-of-focus light from above and below the plane of focus. For live cell imaging, a temperature correction collar allows correction for temperature-induced changes in refractive index of immersion oil that could otherwise produce spherical aberration.

MICROSCOPE CONFIGURATION:

Not applicable. The most important parameters in maximising resolution in the microscope system are the quality of the optical system and correct set-up and alignment of the system.

RECOMMENDED SYSTEM:

Nikon's CFI60 infinity-corrected optical system combines high N.A.s and long working distances in its objectives - providing high-resolution optics for every application.

LINKS:

CFI60 infinity optics: [microscopyu] 

N.A., working distance and other properties of objectives: [microscopyu] 

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