3. Diffraction and Optical resolution


Resolution is defined as the ability of an optical system to differentiate between two closely spaced lines. In terms of the optics, the limit of resolution of a microscope objective refers to its ability to distinguish between two closely spaced Airy disks in the diffraction pattern. The Airy disk represents the absolute limit of resolution achievable by an optical system. (Ultimately, any image is made up of an astronomical number of Airy disks). According to the Rayleigh Criterion, the objects will be resolved when they have a separation of at least R.  In an ideal optical system, the optical resolution is restricted by the numerical aperture (NA) and by the wavelength of light;


Resolution is proportional to the size of the NA and inversely proportional to the wavelength. Therefore, if the highest possible resolution is to be achieved, then either the probing frequency must be increased, or the size of the NA should be increased. Illumination with with ultraviolet light (UV), rather than photopic wavelengths, will permit finer detail, allowing the user to observe better resolution of the subject. However, by decreasing the illumination wavelength, finer and finer detail can be resolved, until a threshold limit is reached, called the diffraction limit. The ideal diffraction limit of an optical microscope is around 200 nm, around half the wavelength of illumination. However, this is an ideal measure which can never be achieved due to imperfections in the optics.

There are two parameters which define the resolution of a digital image, ie. the amount of information that a digital image holds. Firstly, the spatial resolution, which is the number of pixels used to construct an image. Obviously, the more pixels that are present in a given amount of area, the higher the spatial resolution will be. But resolution is also dependent on the dynamic range available to the pixel elements. The dynamic range is the range of intensity values or grey values that the detector can differentiate. The pixels in a digital image will have discreet grey levels that cycle between varying levels from white to black. The cycle rate between black and white levels is called the spatial frequency. When the sampling frequency is reduced, fine details are lost, resulting in aliasing. The greater the dynamic range of a detector, the better the contrast will be. The contrast …..

The resulting image resolution is a measure of the degree to which the digital image represents the fine details of the analogue image recorded by a microscope.

The aspect ratio is the width to height ratio of an image.

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