550 Microscopy /29 : 7 



Dark-field, phase-contrast, interference-contrast, and polarizing micro- 

 scopes have all made it possible to observe living cells in the microscope 

 without staining. These techniques have shown that the structures 

 seen in stained preparations were almost all real and not artifacts. They 

 also have shown that most structures of size greater than 0.2 fx had been 

 seen with the stained preparations. However, the various modifica- 

 tions make more rapid, easier measurements possible, as well as allowing 

 one to observe directly the course of physiological changes in single cells. 



7. Ultraviolet and X-ray Microscopes 



Referring to Equation 5, it can be seen that one way of increasing the 

 resolving power (that is, decreasing b) is by decreasing A . Wave- 

 lengths of electromagnetic radiation just shorter than visible are called 

 ultraviolet and those quite a bit shorter are called X rays. If one is willing 

 to use a photographic image in place of direct visual observation, it is 

 perfectly feasible to build ultraviolet and X-ray microscopes. In theory, 

 they should be able to resolve smaller distances b than can the visual 

 bright-field microscope. <[n practice, the ultraviolet and X-ray micro- 

 scopes are very useful for microspectrophotometry but have not led to 

 better resolution than the visual light microscope. 



The ultraviolet microscope is limited primarily by the problem of 

 focusing. In order to gain an increase in the theoretical resolution of 

 a factor of two, one must go to wavelengths around 200 m/x. The image 

 must first be formed on a fluorescent screen in order to focus the micro- 

 scope, and then a photograph must be taken. To get a reasonably 

 good photograph, it is necessary to have the focus very close to perfect. 

 Any small deviation from exactly proper focusing leads to a loss in 

 resolution; a factor of two or more is almost always lost. 4 



In spite of this the ultraviolet microscope, when used as a micro- 

 spectrophotometer, has played a very important role in the developing 

 analytical picture of the living cell. Perhaps its greatest importance 

 arises from the unique characteristic absorptions of the nucleic acids 

 DNA and RNA. The ultraviolet microspectrophotometer has been 

 used to show that all the DNA is in the chromosomes in dividing cells 

 and that the RNA is distributed throughout the nucleus and the cyto- 

 plasm in living cells. The ultraviolet microscope can also be used to 

 locate a wide variety of other important biological compounds whose 

 characteristic absorption spectra are in the ultraviolet. 



X-ray wavelengths are shorter than ultraviolet, shorter even than 



4 The use of reflection systems in place of lenses allows focusing with visible 

 light and then use in the ultraviolet, thereby reducing the difficulties of focusing. 



