Position referencing in optical microscopy thanks to sample holders with out-of-focus encoded patterns.

This article introduces smart sample holders for optical microscopy. Their purpose is to allow the absolute determination of the position of the observed zone with respect to the sample holder itself and with a high accuracy. It becomes then straightforward to find a given zone of interest by positioning coarsely the microscope slide to the same position coordinates. Furthermore images recorded during different observation sessions; i.e. for slightly different positions; can be processed numerically in order to superimpose them with a high accuracy. Thus the slight deviations of the microscope slide position and orientation due to the different observations are compensated numerically and a perfect superimposition of the recorded images is performed. Then accurate site-by-site image comparisons become possible even for images recorded during different observation sessions and over a long period of time. The subpixel capability of the proposed method is demonstrated and those smart microscope slides constitute a new tool for live cell experiment. In practise, an encoded geometrical pattern used as position reference is inserted in a plane parallel to the surface receiving the tissue section or sample. Then the transition of the focus position from the tissue section to the position reference requires only a vertical adjustment and does not affect the lateral coordinates of observation. The numeric processing of the image of the position reference pattern allows the retrieval of the lateral coordinates that are also used for the tissue section image. Thus each image is recorded with a set of position coordinates that defines accurately the position of the observed area with respect to the sample holder itself.