Cholesterol is a very crucial lipid for animal cells, since it is the building block of cellular membranes, control fluidity of membranes, regulate intracellular transport, cell signalling, nerve conduction and serve as a precursor for synthesis of vitamin D and steroid hormones. Cholesterol is such an important biological molecule for animal cells, yet it has been a real challenge so far, to understand distribution of cholesterol within the biological membranes. In our study, using ultra-high resolution ion microprobe (NanoSIMS) imaging enables us to detect incorporated cholesterol in cells, introduced as 13C labeled cholesterol, in nanometer scale. Then, we are able to monitor the localization of detected cholesterol in cellular structures by combining NanoSIMS (Secondary Ion Mass Spectrometry) with electron microscopy. Our aim in this study is to determine level of incorporated cholesterol in cellular membranes, secretion and endocytic systems, by the help of NanoSIMS imaging combined with electron microscopy.