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Microscopy ..... DF, Rheinberg, Polarization ..... Phase, DIC, Fluorescence ..... Electronic Enhancement

DIC, Phase Contrast, and Fluorescence

Phase Contrast & DIC Illumination

Contrast is, perhaps, the single most important variable in obtaining a good microscope image. Organelles of cells have little inherent difference in light absorbance, so methods must be used that take advantage of other differences. Darkfield (previous page) is a simple method of adding contrast to organelles with differing refractive indices. Phase Contrast and Differential Interference Contrast (DIC) are more elegant optical systems that work at higher magnifications.

 

Phase Contrast: The illumination for phase contrast is by means of a hollow cone of light. Unlike darkfield, however, this technique requires a specialized microscope objective that includes a "phase annulus" - a ring-shaped coating on one of the lens elements - that must match the incoming cone. Phase contrast greatly increases the apparent contrast between cell organelles. As seen in the prostate cancer cell (right) the phase contrast background is a mid-line gray, phase-dense organelles appear dark, and there is a bright halo around the cell.

 

DIC image exampleDifferential Interference Contrast: Most involved - and perhaps the most useful because of its high resolving power - is the method called Differential Interference Contrast (DIC). This technique requires polarization filters - a polarizer and an analyzer - but it also requires additional prisms on either side of the specimen. One beauty of DIC is that images have a three-dimensional appearance with which we are most familiar in the real world. The technique allows the addition of "pseudo-color" to further improve visual contrast between organelles.

 

Fluorescence

flourescence diagramOne elegant method of improving the contrast between organelles is by fluorescence microscopy. Fluorescence is the property of a molecule illuminated with one wavelength (color) of light to emit a second, longer wavelength of lower energy. An example is fluorescent paint that glows when illuminated by a "black light".

 

 

 

A number of fluorescent probes have been developed that can be used to label specific organelles, surface structures, live versus dead bacteria (below, left) and even ions that move into and out of the cells.

live vs. dead bacteria fluorescence
fluorescent stain image

An endothelial cell (above, right) from the bovine pulmonary artery has been labeled with three different fluorescent stains. The nucleus fluoresces blue, the microtubules that form the cytoskeleton fluoresce green, and mitochondria are red.

 

 

 

 

 

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