Fluorescence microscopy

When a fluorescent sample is illuminated with light of the proper WaveLength (in the absorption spectrum of the fluorescence molecules), it will emit light of a longer wavelength. This emitted light can then be detected using, for example, a CCD camera. A camera will acquire a two dimensional image of the emitted light intensity. The light emitted from out-of-focus regions cannot however be distinguished from the light emitted from the in-focus light. Therefore the image will be a combination of a sharp, focused image of the in-focus plane with a blurred, unsharp image of the out-of-focus light. (It is for this reason that the primary application of a Wide Field Microscope is in the imaging of thin samples). 1

An improvement to reject this out-of-focus light is the use of a pinhole, as in a Confocal Microscope . In any case, the acquisition of several 2D planes will build a volume representation of the object, a Three Dee Image .

See Confocal Microscope and links therein for more details. See also Microscope Type .


1 Image Restoration in Fluorescence Microscopy, G.M.P. van Kempen. Delf University Press, 1998. (ISBN 90-407-1792-3 / CIP)

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