Testing mounting media to eliminate background noise in confocal microscope 3-D images of insect genitalia

Recently, the confocal laser scanning microscope (CLSM) has been used to image and generate three-dimensional reconstructions of miniscule insect tissues and cuticular structures. These three-dimensional reconstructions provide the investigator with key information concerning the spatial relationship among structures and substructures. Unfortunately, there can be high levels of background "noise" which can obscure the specimen in these three-dimensional reconstructions. This background "noise" might be a result of the mounting media either autofluorescing or reflecting and scattering the imaged specimen's fluorescence. The standard nonpermanent mounting medium is glycerine jelly (a 1:17:17 ratio of porcine gelatin to glycerine to water). In this study, the organic molecule (lipid, protein, or carbohydrate) added to the glycerine water mixture was varied. The relative background to specimen signal (the mean voxel brightness reading in ImageJ freeware) was compared across mountants. The mounting media tested are ranked from best (least background noise) to worst (most background noise) as follows: agarose, agar, pectin, gelatin (the standard), petroleum jelly. A 1% agarose mountant (1:50:50 ratio of agarose to glycerine to water) is recommended because it causes little to no background noise, provides consistent high quality contrast between specimen and background with increasing depth, and is easy to handle.