Welcome to Phase-Contrast-Microscope
First described in 1934 by Frits Zernike is the phase contrast microscopy. The phase contrast microscopy is a contrast enhancing optical technique that can be utilized to produce high contrast images of transparent specimens, like living cells, microorganisms, thin tissues slices, lithographic patterns, fibers, latex dispersions, glass fragments and sub cellular particles. In effect, the phase contrast technique employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. Living cells can be examined in their natural state without previously being killed, fixed, and stained is one of the main advantages of phase contrast microscopy. As a result, the dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail.
Partially coherent illumination produced by the tungsten halogen lamp is directed through a collector lens and focused on a specialized annulus positioned in the sub stage condenser front focal plane. Illuminates the specimen and either passes through undeviated or is diffracted and retarded in phase by structures and phase gradients present in the specimen when wave fronts passing through the annulus. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase contrast image observed in the eyepieces. Prior to the invention of phase contrast techniques, transmitted bright field illumination was one of the most commonly utilized observation modes in optical microscopy, especially for fixed, stained specimens or other types of samples having high natural absorption of visible light. Specimens readily imaged with bright field illumination are termed amplitude objects because the amplitude or intensity of the illuminating wave fronts is reduced when light passes through the specimen.
The addition of phase contrast optical accessories to a standard bright field microscope can be employed as a technique to render a contrast enhancing effect in transparent specimens that is reminiscent of optical staining. Light waves that are diffracted and shifted in phase by the specimen can be transformed by phase contrast into amplitude differences that are observable in the eyepieces. Easily visualized with phase contracts optics due to diffraction and scattering phenomena that occur at the edges of these objects are large, extended specimens. The performance of modern phase contrast microscopes is so refined that it enables specimens containing very small internal structures, or even just a few protein molecules, to be detected when the technology is coupled to electronic enhancement and post-acquisition image processing. The cells are human brain tissue grown in monolayer culture bathed with a nutrient medium containing amino acids, vitamins, mineral salts, and fetal calf serum. The cells appear semi transparent with just highly refractive regions in bright field illumination. When observed using phase contrast optical
accessories, the same field of view reveals significantly more structural detail. Cellular attachments become discernable, as does much of the internal structure. The contrast range is dramatically improved.
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