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Glossary of Light Microscopy Terms

Binning

Binning is a process used to increase the sensitivity of a CCD camera by combining the light-sensitive wells (i.e.pixels) together to boost the signal. If we imagine a hypothetical camera (see below) with a chip consisting of 4 x 4 pixels and we apply a binning factor of two then 2 x 2 arrays of adjacent pixels will be combined together to form an array of 'super pixels' that are four times the area of the originals and contain the combined signals of the adjacent pixels. This improves the sensitivity because CCD camera readout noise often obscures low signals. By combining the signal from adjacent pixels the readout noise threshold can be exceeded.

The drawback is that the resolution is lower because the specimen is now being sampled (in the example above) by four large pixels instead of 16 small ones. The images below broadly illustrate the effect of increasing binning factors on resolution. In the high resolution imaging modes used in fluorescence microscopy you would have to use binning factors higher than 2 to experience the pixelation seen in the images on the right.

Numerical Aperture (NA)

The numerical aperture is a dimensionless value that characterises the resolution of a lens. It is defined by the following equation:

NA = n sinθ

Where n is the refractive index of the immersion medium and θ is the half-angle of acceptance of the lens.

The higher the NA the higher the resolution of the lens. The NA cannot exceed the refractive index of the immersion medium, so lenses for use in standard immersion oils (n ≈ 1.51) tend to have a maximum NA of 1.4 to 1.45

Links:

Numerical Aperture (Wikipedia)

Working Distance (WD)

The working distance is the distance from the front of the lens to the nearest surface of the coverslip when the specimen is in sharp focus. For objective lenses designed for use without a coverslip the surface of the culture vessel or specimen in used.

Links:

Working and Parfocal Distances (microscopyu)

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