Position-referenced microscopy (PRM) is based on intelligent sample holders that integrate a position reference pattern (PRP) in their depth, allowing the determination of the lateral coordinates with respect to the sample-holder itself. compatible with the working distance of the lens. A photograph of a smart culture dish that has been developed is shown in Fig. 2. The PRP is first produced by photolithography on a glass coverslip and then covered by the transparent polymer layer. This position-referenced part is finally stuck on the bottom side of usual culture dish that has been previously pierced to minimize the final thickness of the smart culture dish obtained. The encoded surface is 1phase ambiguities inherent to phase measurements are compensated in an absolute way through the decryption of the missing dot distribution present in each field of observation. Once a local image of the PRP is recorded, its position with respect to the PRP coordinate system is reconstructed in two steps. The first step involves linear phase processing. It allows the sharp localization of the periodic dot distribution with respect to the pixel frame of the recorded image (fine measurement) as well as the in-plane orientation of the view. The second step involves binary image processing. Its aim is to identify the distribution of the missing spots for the determination of the order of the lines and columns under view. This results in a coarse but absolute position measurement. The latter is combined with the fine but relative measurement provided by the phase processing to finally give the fine and absolute position of the zone under view. 3.2. Effect of PRP on light budget If an inverted microscope is R547 used as described in Fig. 1, the PRP has to be crossed twice in the formation of the biological material image. This section considers the impact of the PRP on the imaging system performance and on the final light budget of PRM. From the point of view of optical wave propagation, the problem consists in determining the disturbances of the point spread function (PSF) observed in the focusing plane that are due to the presence of the PRP. Figure 3(a) represents the intensity distribution of the converging wave in the plane just behind the PRP. In this plane, the optical wave is given by the product of the PRP transparency by usual spherical wave, here with a gaussian intensity distribution. The phase change due to the PRP Slc4a1 comes from the coverslip thickness and can be considered to be uniform. The converging wave has thus usual spherical phase distribution associated with an intensity distribution as in Fig. 3(a). The propagation of the altered spherical wave up to the focusing plane has been calculated by using the angular spectral range of aircraft waves strategy in the scalar approximation [14, 15, 17]. A 2D Fourier transform can be first put on the converging beam in the aircraft simply behind the PRP. The angular spectral range of plane waves is obtained thus. The latter can be after that propagated up to the concentrating aircraft by applying the correct stage adjustments. Finally, an inverse 2D Fourier transform is conducted to get the preferred PSF. Open up in another windowpane Fig. 3 PRP effect on PSF: (a) converging influx after PRP crossing; (b) computed PSF in the concentrating R547 aircraft in logarithmic size. Computations have already been performed on R547 generated pictures of 2048 2048 pixels digitally, having a sampling range of 0.2that avoids aliasing, as well as for a wavelength of 0.45 2with a period of 4as experimentally used. Different proportions of absent dots have already been considered to be able to assess the aftereffect of this parameter for the light spending budget. In Fig. 3(a), just.