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The standard two-dimensional (2D) image recorded in bright-field fluorescence microscopy is rigorously modeled by a convolution process involving a three-dimensional (3D) sample and a 3D point spread function. We show on synthetic and experimental data that deconvolving the 2D image using the appropriate 3D point spread function reduces the contribution of the out-of-focus fluorescence, resulting in a better image contrast and resolution. This approach is particularly interesting for superresolution speckle microscopy, in which the resolution gain stems directly from the efficiency of the deconvolution of each speckle image.

Numerical approach for reducing out-of-focus light in bright-field fluorescence microscopy and superresolution speckle microscopy