Fluorescence and Phase Contrast Microscopy

In the needle with width 300 nm (1), diameter of a thin needle 300 nm (2) and of a thick needle 500 nm (3). (c) Detail of break 1 (image size 5x4 pm ). 

As can be seen, it is possible via fluorescence microscopy to investigate structures with characteristic dimensions of a few hundred nanometers. In order to determine the real dimensions, however, complementary methods such as atomic force microscopy are necessary. 

It has to be noted that, due to Abbe s diffraction limit, two objects cannot be individually observed which are separated by less than A/2. However, if there is only a single object within the resolution limit the position of it can be determined with much higher precision. For this one has to scan spatially over the object, measuring the spatial intensity distribution. The precision of determining the position, 5x, is then approximately given by 

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Fig. 7. Histochemical localization of MM-CPK in the M-line region of the sarcomere of an isolated chick muscle fiber. Localization is by means of reaction of a fluorescent anti-MM-CPK antibody with the enzyme. Fluorescence and phase contrast microscopy are superimposed. M, M-line region and Z, Z-line region. By courtesy of H. Kuhn and T. Wallimann of this laboratory.

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