A Method for Illumination of Chorioallantoic Membrane (CAM) of Chicken Embryo in Microscope

This article presents the study and recommendation of a method to improve the observation, in laboratory experiments, using a microscope stereoscopic or stereomicroscope with chicken embryo chorioallantoic membranes (CAM). In these experiments, tumor cells are implanted in this CAM so that they will form a tumor during the embryo's development period. The number of neovessels that develop around the tumors are indicators of results on testing with antiangiogenic drugs. These studies require quantification of vascularization to compare the stimulating or inhibitory effects caused by different agents. The proposed method to improve the visualization of the vessels consists of a lighting chamber composed of three lighting rings, to accommodate the egg, for observation in a microscope. Each ring is composed of sets of 8–16 LEDs, with a temperature of color between 3000 and 5500 K, aligned, focusing on the regions above, central and below the egg. The method provided an improvement in the results as more details of the images were observed, when compared to the images obtained with the original illumination of the adopted microscope stereoscopic or stereomicroscope, model LEICA ZOOM™ 2000. Also, the proposed LED lighting avoids the heating irradiated by the original illumination, which may cause discomfort, dehydration and unnecessary movements of the embryo, besides reflection or glare on the exposed membranes and liquids of the egg, which may impairment the visualization. Qualitative evaluation of sample images demonstrated that subtle vessels stand out, thus allowing better results for further quantitative analysis of the CAM. The quantitative analysis of the images, using the vessel counting method based on a grid, resulted in a mean percentage difference counting of +56% vessels in relation to the counting with the original illumination of the microscope.