FLUORESCENCE MICROSCOPY 101 



Metcalf and Patton (1944) suggest the use of a drop of water or 

 petrolatum on the top of the condenser to serve as a connecting 

 fluid between condenser and slide in order to obtain illumination of 

 high intensity when objectives of twenty times magnification, or 

 higher, are used. With low-power objectives, they point out that it 

 is necessary to remove the top lens or lenses of the condenser so 

 that the field can be properly illuminated. Other investigators have 

 employed sandalwood or Shillaber oil between the slide and con- 

 denser. 



The specimen is mounted on a slide (S) made of an ultraviolet- 

 transparent glass such as the Corex D slide of Corning Glass Co. 

 However, Metcalf and Patton ( 1944) have found that ordinary 

 glass slides of 1.2 to 1.5 mm. thickness may be used when the inten- 

 sity of the ultraviolet radiation is great. A nonfluorescing medium 

 must be used for moimting the specimen; glycerol or mineral oil 

 was recommended by Simpson ( in Cowdry, 1943, pages 76-78) ; but 

 Popper (1944) reported a disturbing fluorescence from glycerol 

 (although others beside Simpson have found no difficulty with it) 

 and the use of mineral oil is limited to substances that will not 

 dissolve in it, e.g., for vitamin A studies Popper ( 1944) used water 

 as the mounting medium. In some instances, petrolatum serves as 

 a good temporary mount, and, for permanent mounts, isobutyl 

 methacrylate (du Pont), suggested by O'Brian and Hance (1940), 

 is probably the best. With immersion objectives, sandalwood or 

 Shillaber oil may be employed as the immersion medium. No special 

 objectives or oculars are required; however, Jenkins (1937) has 

 pointed out that some of the older objectives contain balsam that 

 gives rise to its own fluorescence, and in these cases a darkfield 

 stop must be used in the condenser to prevent the entrance of direct 

 ultraviolet rays into the objective. 



A filter (EF) that excludes ultraviolet, and passes visible rays, 

 is placed on the ocular. Either the Corning filters No. 3389 or 3060, 

 the Leitz No. 8547A, the Bausch and Lomb or the Zeiss Euphos 

 filter, or a circle of Wratten 2A gelatin filter cut to fit inside the 

 eyepiece may be used. INIetcalf and Patton (1944) recommend a 5% 

 solution of sodium nitrite contained in a plane-sided glass cell 5-10 

 mm. in optical depth which may be conveniently placed on the dia- 

 phragm of the ocular or, better still, on the diaphragm of the 

 microscope tube. 



