iiicro:13 



Fluorescence microscopy has been applied in phytonematoloEy as a tech- 

 nique to distinguish between live and dead nematodes based on whether or 

 not they took up a fluorescent stain such as acridine orange. 



Perhaps, the most fertile application of the fluorescence technique would 

 be in intravital microscopy to observe locations and changes of certain 

 materials within plant and animal cells, Some authorities believe that 

 fluorescence microscopy is only Just beginning to be developed and has 

 great promise as another valuable research tool. 



Reflection Objectives 



The final specialized aspect of microscopy, as applied to phytonematology, 

 has to do with the problem of viewing the nematodes at fairly high magni- 

 fications (the higher, the better) in action on plant surfaces, in their 

 soil environment, or on special substrates. The problem which arises in 

 using achromatic and apochromatic objectives ranging in magnification 20X 

 to 90 or lOOX is due to their having rather limited working distances. 

 VJorking distance is defined as the distance from the bottom surface of 

 the objective mount to its focus point or, in other words, the araoiint of 

 clearance that exists between the bottom of the objective and the speci- 

 men. The kinds of observations we would like to make of the nematodes 

 require lenses that permit use of simple micro-observation chambers in 

 which anple illumination of the specimen preferably from above, is possi- 

 ble because of opacity of the plant parts under study or of the substrate 

 used in the chamber. As pointed out in the beginning of this talk, the 

 use of lens systems of the optical design, as those found on stereoscopic 

 dissecting microscopes, is not satisfactory. Although these lenses do 

 provide ample working distance, the resolution and the highest magnifica- 

 tions are not sufficient for detailed feeding studies and the like. 



There is now commercially available a "new" type of microscope objective 

 referred to as the reflection objective. Such objectives overcome the 

 problems of getting ample working distance so that focusing deep into a 

 micro-observation chamber with adequate incident illumination is possible, 

 even at reasonably high magnifications. 



The development of reflecting objectives is really not new. However, it 

 is not generally known among those who now daily use the microscope that 

 in the early days of this instrument both reflecting and refracting 

 objectives x^rere developed and improved and that instruments utilizing 

 both types of lenses were put to practical use. The development of the 

 achromatic objective around I82I4 and its subsequent improvements more or 

 less made the reflecting objective obsolete. It is fortunate that inter- 

 est and progress in the further development of the reflection microscope 

 has been revived, thanks particularly, I think, to the ultra-mi croanalyti- 

 cal chemists. 



Two types of reflecting objectives are available. In one (Figure Ua ) , 

 the metallic coated reflecting surface or mirror is such that an image of 



