Method of forming an imaging reference device

A method is provided of forming an imaging reference device (or calibration standard) 10 for a biological material imaging system. A first region is provided in a substrate formed from an optically transmissive material which is an analogue of biological tissue. This first region is stained using a first biological tissue stain. A second region is provided in a second substrate which is optically transmissive and an analogue of biological tissue. The second region is stained with a second biological tissue stain. The first and second regions are overlapped as a pair, such that light incident upon the pair is modulated by the respective first and second biological tissue stains of the respective regions. Multiple regions of the first and second type may be provided, stained to different degrees. The substrates may be formed from a material such as cellophane (RTM), cellulose, agar, agarose or gelatine. The stains may be those used for the Haematoxylin and Eosin (H&E) staining protocol.