Dark-field microscopy technique

At this point it is worth comparing the different techniques of contrast enliancements discussed so far. They represent spatial filtering teclmiques which mostly affect the zeroth order dark field microscopy, which eliminates the zeroth order, the Schlieren method (not discussed here), which suppresses the zerotii order and one side band and, finally, phase contrast microscopy, where the phase of the zeroth order is shifted by nil and its intensity is attenuated. 

Dark field Visualization technique for ashes produced by microincineration and fluorescence microscopy useful for low-contrast subjects Electron systems imaging EM shadowing Detection, localization, and quantitation of light elements Structural information from ordered arrays of macromolecules... 

In dark-field microscopy, the particles are only a blur no details are distinguishable at all. Some rough indication of the symmetry of the particles is afforded by the twinkling that accompanies the rotation of asymmetrical particles, but this is a highly subjective observation. However, the technique does permit the rate of particle diffusion to be observed. We see in Chapter 2 how to relate this information to particle size and shape. The number of particles per unit volume may also be determined by direct count once the area and depth of the illuminated field have been calibrated. This is an important technique for the study of coagulation kinetics, a topic we discuss in Chapter 13. 

Two techniques for overcoming the limitations of optical microscopy are of particular value in the study of colloidal systems. They are electron microscopy36-37, in which the limit of resolution is greatly extended, and dark-field microscopy, in which the minimum observable contrast is greatly reduced. 

Dark-field electron microscopy (in which the image is formed from the scattered beam), when combined with improved techniques of sample handling and preparation and minimal radiation exposure, can lead to images of sufficiently undamaged DNA at a resolution of 10 A (116). Figure 45 shows such an image in which the two-dimensional projection of the helix is clearly visible on the undamaged part of the molecule. 

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