Ultraviolet microscopy 



BASIC PRINCIPLES AND DESIGN 



The wavelength region of the visible spec- 

 trum is 400 to 700 m/i. The adjacent shorter 

 wavelength region (100 to 400 m^) is the 

 (invisible) ultraviolet region. Below 100 niM, 

 air absorption restricts the transmission of 

 ultraviolet, and this region is called the 

 "vacuum ultraviolet" region. 



Inasmuch as resolving power is pro- 

 portional to wavelength, it is possible to 

 resolve finer structure in ultraviolet than in 

 visible radiation. Also, since many biological 

 materials have absorption bands in the ultra- 

 violet, their microstructure may, by the use 

 of the ultraviolet microscope, be rendered 

 visible without resort to staining. 



The ultraviolet microscope differs from 

 the conventional light microscope in that it 

 includes means for converting the invisible 

 ultraviolet unage into a visible image. 

 Furthermore, since optical glasses do not 

 transmit ultraviolet, other materials, notably 

 fluorite and fused quartz, are used for lens 

 elements, and mirror systems often are used 

 in place of refracting optics. These same 

 differences also apply to the illuminating 

 system, and of course, the source itself is 

 different, since it must emit in the ultra- 

 violet rather than the visible, and cannot be 

 enclosed in a glass envelope, since glass ab- 

 sorbs the ultraviolet. 



Conversion of the ultraviolet to visible 

 energy is accomplished by (1) photography, 

 (2) fluorescence, (3) television type receptor 

 tubes, (4) flying spot television techniques, 

 or (5) photoemissive image-converter tubes. 

 Early History. Koehler (1) developed 

 the first optical systems for ultraviolet 

 microscopy in about 1904, his work being 

 aimed toward the goal of increased resolving 

 power due to the use of shorter wavelengths. 

 Microscopes made in accordance with 

 Koehler's design were made by the Zeiss 



firm The objectives were monochromats, 

 i.e., corrected for only one wavelength in 

 the ultraviolet. Photography was used to 

 render the image visible. Work with this 

 instrument was difficult, and apparently 

 very little real application was made of it. 



Spectral Absorption. In the 1930's, in- 

 terest in ultraviolet was revived by the work 

 of T. Caspersson (2, 3) in Sweden, and J. 

 Loofbourrow (4, 5) in the United States, in 

 studying the spectral absorption properties 

 of various biochemicals. With ultraviolet, 

 these men found that it was possible to 

 photograph live unstained tissue sections 

 and tissue cultures, and to differentiate vari- 

 ous normal and abnormal cells by their 

 absorption characteristics. 



Objective Designs. Studies of this nature 

 led to a re-activation in the field of ultra- 

 violet microscope design improvement. L. V. 

 Foster (6), described in 1946, an achromatic 

 objective composed of fused ciuartz and 

 fluorite for use in a sufficiently broad range 

 in the ultraviolet, so that a fluorescent screen 

 could be used for focusing. Interest in spec- 

 tral absorption studies, however, led to the 

 demand for still greater wavelength range, 

 and mirror type objectives were designed 

 by Brumberg (7), Burch (8), and Grey (9) 

 to fulfill this requirement. 



The most widely used of these are the 

 Grey designs, which employ a combination 

 of reflecting and refracting optics to achieve 

 good correction over a large wavelength 

 range with very little central obscuration by 

 the mirror system. The refracting elements 

 are of fused quartz and fluorite Fig. 1, shows 

 the construction of the 0.72 N. A. Grey 

 design as made by Bausch & Lomb. This 

 objective is corrected for the complete 

 spectral range from 254 mju to 700 m/x, hence 

 is useful in applications where focusing is 

 accomphshed in the visible for photography 

 in the ultraviolet. 



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