A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

Abstract

In this work, for the first time, the quality of restoration in wide-field microscopy images after deconvolution was analyzed as a function of different Point Spread Functions using one deconvolution method, on a specimen of known size and on a biological specimen. The empirical Point Spread Function determination can significantly depend on the numerical aperture, refractive index of the embedding medium, refractive index of the immersion oil and cover slip thickness. The influence of all of these factors is shown in the same article and using the same microscope. We have found that the best deconvolution results are obtained when the empirical PSF utilized is obtained under the same conditions as the specimen. We also demonstrated that it is very important to quantitatively check the process' outcome using several quality indicators: Full-Width at Half-Maximum, Contrast-to-Noise Ratio, Signal-to-Noise Ratio and a Tenengrad-based function. We detected a significant improvement when using an indicator to measure the focus of the whole stack. Therefore, to qualitatively determinate the best deconvolved image between different conditions, one approach that we are pursuing is to use Tenengrad-based function indicators in images obtained using a wide-field microscope.