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.

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