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Contrast | Contrast

Key Words: fluorescence, DIC, darkfield, FLIM, Resolution, brightfield, phase contrast, oblique illumination, Noise Terminator, filter block, signal to noise, polarized light

Definition:The difference between an area of interest and the background in an image defined by differences between the brightest and darkest point in an image or by differences in color


Many subjects in microscopy have very low inherent contrast. Living cells, for example, are largely translucent and not strongly colored making it difficult to distinguish specific features. Creating contrast in a specimen enables the observer to identify key structures. This can be achieved either through the manipulation of microscope illumination or by the addition of probes targeted at specific features. Examples of illumination techniques to create contrast include darkfield, phase contrast, oblique illumination, DIC and polarized light. These take advantage of differences in refractive indices, relief, and anisotropy between different parts of the specimen. Probe techniques include the use of targeted histological stains to create color contrast and fluorescent probes to 'light up' key molecules and structures when excited with light of the appropriate wavelength. In fluorescence imaging, filter blocks are used to (ideally) allow only selected wavelengths of light to reach the detector so that fluorescence light is contrasted against a black background. Stray light in the system can reduce this contrast and compromise imaging especially in situations where fluorescence emission is weak. Several fluorescent probes with different emission spectra can be used to identify multiple structures in a specimen as long as emissions can be cleanly differentiated. Overlapping spectra may be resolved either through spectral imaging or by the use of fluorescence lifetime as a contrast parameter.


Illumination techniques are useful in live cell imaging where they allow cell features to be observed without the need to used potentially toxic stains or fluorophores. Illumination-derived contrast is also important in industrial imaging enabling precise measurement of surface features and in distinguishing properties of crystalline materials (polarizing microscopy). Probes in the form of targeted histological stains are predominantly used in fixed biological specimens. Fluorophore probes, especially the genetically encoded proteins such as the GFPs provide a powerful means of generating contrast in living cells. Multiple probes may be viewed simultaneously in one specimen to provide information about the spatial relationships between molecules of interest (especially when using confocal imaging).


This is dependent on the application and imaging technique being used.


The modular design of the Ti inverted microscope system allows users to exploit a variety of contrast and imaging techniques on one microscope such as epifluorescence, DIC, phase contrast, and Hoffman Modulation Contrast. This system also integrates Noise Terminator technology to dramatically improve contrast in fluorescence imaging.


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