A simple and low-cost method to visualize musculature and other aspects of anatomy by confocal microscopy

Confocal microscopy study of musculature and other anatomical structures in whole-mount preparations of arthropods and some other cuticle-bearing animals often presents a significant difficulty because the cuticle poses a barrier to fluorescent dyes and their pigmented tissues can cause attenuation of fluorescent signal. This paper describes a simple and inexpensive procedure based on the use of clove oil as a tissue-clearing, staining, and mounting medium that helps overcome the problem of slow dye penetration and tissue opaqueness and allows muscles and several other organ systems to be visualized by confocal or epifluorescent microscopy. This clove oil-induced fluorescence (COIF) method relies on the ability of clove oil to accumulate in muscles and some other tissues and become steadily fluorescent if irradiated at 488 nm. For this method, animals were fixed in 70% ethanol or 4% formaldehyde, then dehydrated and mounted in clove oil. Heavily pigmented animals were additionally bleached in hydrogen peroxide prior to the dehydration step. The COIF method showed excellent results in all major groups of arthropods and some mollusks and annelids revealing the three-dimensional arrangement of muscles, gonads, glands, cellular nuclei and some parts of the nervous and digestive systems. In the other animal groups tested, clove oil stained all tissues making it difficult to observe the anatomical details. The COIF method is advantageous in some respects over other methods such as phalloidin staining because of its tissue penetration and clearing abilities, low cost of the reagents, resistance to photodamage and the possibility of staining museum specimens.