Deconvolution and TILLvisDECO package

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Introduction

Wide-field fluorescence microscopy can be used to acquire images (2D and 3D) of sub-micron resolution. However, convolution and out-of-focus effects blur the resulting images, reducing the ability to resolve the finer details of biological structures. These are the result of several factors:

Cells before Deconvolution Cells after Deconvolution

• The image plane is convolved by the finite optical aperture in a way that can be described by diffraction theory. The diffraction limited resolution of the microscope is related to the wavelength of the light used, the numerical aperture (NA) of the lens and the refractive index of the milieu. This leads to the Rayleigh criterion that defines the minimum resolvable distance between two distinct points in two dimensions.
• Blurring due to slight positional errors in the focal distance to the imaging plane result in further convolution of the image. Blurring or noise is also caused by objects with low spatial frequency (i.e. objects which have a large depth of field) that contribute light to the image from neighbouring focal planes above or below that in focus. This results in fine details becoming smoothered in "out of focus light" and thus lost, or at least visualized with much reduced contrast.
• Optical aberrations can also contribute to a degradation of image quality, which they affect in three dimensions. Aberrations are classified as five types: spherical, astigmatism, curvature of field, distortion, and coma. However, aberrations are greatly reduced through the use of modern, well-corrected, high quality objectives.