A rapid stain-clearing method for video based cytological analysis of cotton megagametophytes

Optical "clearing" is a cost saving method for preparing large numbers of whole, dissected or thickly sectioned cytological specimens such as plant ovules and ovaries. Minimal labor is required and specimens retain three-dimensional integrity. Previous development of high contrast stain-clearing methods using hemalum to impart contrast has facilitated analysis and photography under bright field illumination for small ovules. The deep stain intensity of hemalum, however, often precludes adequate light transmission and contrast within internal focal planes, limiting the applicability of hemalum-based stain-clearing to small specimens. Having encountered this problem for nucelli of cotton (Gossypium barbadense L.), which are roughly 300 microns thick at fertilization, we have developed a modified stain-clearing system. The two key features of these new methods are the use of azure, C, which allows the intensity of staining to be readily regulated, and contrast manipulation via video signal and image processing. Intensity of azure C stain was readily controlled by modifying the staining and/or dehydration media to produce relatively low contrast specimens. Analysis was facilitated by indirect viewing on a video monitor using adjustments of sensitivity, exposure, and contrast of the charge-coupled device (CCD) camera. Digital processing provided further enhancement. Acceptable images were obtained from virtually all specimens. These methods, which combine low contrast (high transmittance) specimens with high contrast imaging, should facilitate data acquisition on reproduction, thus the developmental and genetic characterization of reproductive mutants. Other applications, e.g., in pathology and embryology, are readily envisioned.