PLUORESCENCE MICROSCOPY 



99 



FLUORESCENCE MICROSCOPY 



ultraviolet. Aluminum and magne- 

 sium-aluminum alloys are best for this. 

 By mounting the microscope and light 

 source horizontally this item can be 

 eliminated. 



5. An ordinary microscope that is 

 fitted with a substage condenser of 

 quartz or ultraviolet transmitting glass. 

 Since the ultraviolet light has served its 

 purpose when it has reached the tissue, 

 ordinary glass objectives and eyepieces 

 are used. With some older objectives 

 the balsam of the lenses fluoresces in 

 ultraviolet and causes an unpleasant 

 diffuse light to appear in the microscope 

 that masks the fluorescence of the tissue. 

 This may be eliminated with a darkfield 

 stop that prevents direct rays of ultra- 

 violet light from entering the objective. 

 Newer lenses are free from this fluores- 

 cence and may be used without a dark- 

 field stop. This is desirable since it 

 permits the utilization of a greater por- 

 tion of the light that strikes the con- 

 denser. Popper lias reported that the 

 fluorescence of Vitamin A can be ob- 

 served with an ordinary microscope with 

 glass condenser. Ordinary optical glass 

 transmits sufficiently far into the near 

 ultraviolet that this type of apparatus 

 might be successfully used for strongly 

 fluorescent substances. 



6. Slides for the specimens of ultra- 

 violet transmitting glass. (Corex D 

 glass slides, obtainable from Corning 

 Glass Co. are suitable.) 



7. An eyepiece filter that excludes 

 ultraviolet light with a minimum ab- 

 sorption of visible light. This may be 

 of glass (Leitz ultraviolet protecting 

 filter no. 8574 A, Corning Glass Works 

 filters no. 3389 or 3060) or, simplest and 

 cheapest, a circle of Wratten 2A gelatin 

 filter cut to fit within the eyepiece (the 

 Wratten 2 filter is not suitable since it 

 fluoresces itself in ultraviolet light). 



8. Non-fluorescent media for mount- 

 ing the section to be examined. Me- 

 dicinal mineral oil, or glycerin is suit- 

 able. If immersion lenses are to be used 

 a non-fluorescing immersion medium 

 must be employed. Sandlewood oil has 

 been recommended for this purpose. 



Preparation of tissues: Hamperl (loc. 

 cit.) recommends that tissues for fluores- 

 cence examinations be fixed only in a 

 dilute solution of formalin, since metal 

 containing fixatives destroy the fluores- 

 cence of some substances. A 5-10% 

 solution of U.S. P. formalin in aq. dest. 

 is ordinarily employed. Tissues should 

 should not be fixed longer than 24 hrs. ; 

 certain components of tissue acquire 

 abnormal fluorescence if the time of 

 fixation is prolonged. If fats and other 

 alcohol soluble substances are to be ex- 



amined, i.e., vitamin A, polycyclic 

 organic carcinogens, etc., frozen sections 

 must be made. If these substances are 

 not of interest, the tissue may be de- 

 hydrated, cleared, and imbedded in 

 paraffin in the usual manner. High 

 quality reagents are required, because 

 the impurities found in many organic 

 substances themselves fluoresce. All 

 paraffin must be removed since this too 

 fluoresces. The section can be cleared 

 in anhydrous glycerin or pure medicinal 

 mineral oil. Gelatin and celloidin are 

 not recommended for imbedding because 

 of their fluorescence. 



Two types of fluorescence may be pro- 

 duced in tissues with this type of appa- 

 ratus. The first is that seen in tissues 

 that have been subjected to no special 

 treatment and is due to the presence of 

 fluorescent substances in the tissues 

 themselves. This is termed "primary" 

 fluorescence or natural fluorescence and 

 is exhibited by many substances found 

 in animal organisms. In most tissues 

 there are present sufficient quantities of 

 these materials to permit the observer to 

 recognize the general structure of the 

 tissue without recourse to stained con- 

 trol sections studied with transmitted 

 visible light. Hamperl (loc. cit.) de- 

 scribes, in considerable detail, the 

 natural fluorescence of many human 

 tissues. Jenkins (loc. cit.) summarizes 

 the findings in the most common animal 

 tissues. Cornbleet and Popper (T.and 

 IL, Arch. Dermat. & Syph., 1942, 46, 

 59-65) review the natural fluorescence 

 of human skin. Popper and his co- 

 workers have contributed a series of 

 papers on the fluorescence of vitamin A 

 in animal tissues (Popper, H., J. Mt. 

 Sinai Hosp., 1940, 7, 119-132. Arch. 

 Path., 1941, 31, 766-802 ; Popper, H. and 

 Brenner, S., J. Nutrition, 1942, 23, 431- 

 443; Popper, H. and Pia,gins, A. B., 

 Arch. Path., 1941, 32, 258-271). Simp- 

 son and Cramer (W. L. and W., Cancer 

 Research, 1943, in press) have used the 

 method to follow the distribution and 

 persistance of methylcholanthrene in 

 skin. 



Another kind of fluorescence is the 

 "secondary" fluorescence that appears 

 in certain components of the tissue after 

 sensitization with dyes and plant ex- 

 tracts. This extends considerably the 

 range of fluorescence microscopy and has 

 been developed chiefly by Haitinger 

 (loc. cit.) in conjunction with Hamperl 

 and Linsbauer. Various fluorescent al- 

 kaloids, azo dyes, primulins, auramine, 

 berberine sulfate, chelidonium, rhubarb 

 extracts, etc., are selectively absorbed 

 by certain parts of the cell and cause 

 them to show characteristic fluorescences 



