Evaluation of a new method for the determination of experimental PSF of a wide-field microscope using white-light and a linear sensor

摘要

In the biology field there is a high demand for three-dimensional (3D) microscopy techniques owing to the need for obtaining depth information. Confocal microscopy is the reference method but it has the drawback of slow image acquisition time. Different configurations were developed to obviate this such as spinning-disk that basically consists in a set of parallel confocal microscopes and line scanning that uses line illumination so the scanning in the fast axis is not required. Amongst other 3D techniques there is the digital microscopy that normally uses a wide-field microscope to acquire 2D images (optical sections) of the specimen containing substantial contributions from out-of-focus portions. To remove this, a computational method is used that is based on the definition of a model for the process of image formation and recording. A compromise should be taken between a more accurate model with best results or a simplified one with a lower computational processing time. This work is based on a laboratory prototype of a wide-field microscope with white-light illumination using a CMOS linear image sensor. It aims at the application of one simplified model that takes advantage of a new method for the determination of experimental point spread function (PSF) that uses a specimen consisting of a set of lines in two-perpendicular directions due to the anisotropy that results from detector geometry. The 1D and 2D PSF were built. A simulation of the application of the model was developed using Matlab using different simulated specimens. Its preliminary results will be presented.