Colour Vignetting Correction for Microscopy Image Mosaics Used for Quantitative Analyses

Abstract

Image mosaicing permits achieving one high-resolution image, extending the visible area of the sample while keeping the same resolution. However, intensity inhomogeneity of the stitched images can alter measurements and the right perception of the original sample. The problem can be solved by flat-field correcting the images through the vignetting function. Vignetting correction has been widely addressed for grey-level images, but not for colour ones. In this work, a practical solution for the colour vignetting correction in microscopy, also facing the problem of saturated pixels, is described. In order to assess the quality of the proposed approach, five different tonal correction approaches were quantitatively compared using state-of-the-art metrics and seven pairs of partially overlapping images of seven different samples. The results obtained proved that the proposed approach allows obtaining high quality colour flat-field corrected images and seamless mosaics without employing any blending adjustment. In order to give the opportunity to easily obtain seamless mosaics ready for quantitative analysis, the described vignetting correction method has been implemented in an upgraded release of MicroMos (version 3.0), an open-source software specifically designed to automatically obtain mosaics of partially overlapped images.

Cauliflower Mosaic Virus TAV, a Plant Virus Protein That Functions like Ribonuclease H1 and is Cytotoxic to Glioma Cells

“…Nuclear proteins were extracted using RIPA Buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) supplemented with Halt Protease and Phosphatase Inhibitor and DNase 1 μg/ml (Qiagen), incubated 30 minutes on ice in agitation, and then spun at 17000g for 15 minutes at 4°C. Nuclear proteins (supernatant) were collected and prepared for western blotting that was performed using the GAPDH-HRP-conjugated antibody (Origene, 2D9) for endogenous cytoplasm marker, anti-TAV rabbit polyclonal serum [27,28], and histone H3 as a control for nuclear contamination.…”

“…e following commercially available antibodies were used in immunofluorescence: polyclonal rabbit anti-CaMV IgG (DSMZ, AS-0206) and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody conjugated to Alexa Fluor 594 (red color; 1 : 600 in blocking solution). TAV antiserum is a noncommercial rabbit anti-TAV polyclonal serum [27,28].…”

Image Vignetting Correction Using a Deformable Radial Polynomial Model

“…The appearance of this effect is particularly undesirable when there is a need for radiometric or quantitative image analysis, which is very common in different areas, e.g., astronomy [ 1 , 2 ]; microscopy [ 3 , 4 , 5 , 6 ]; and remote sensing applications using terrestrial [ 7 , 8 ], airborne [ 9 , 10 , 11 , 12 , 13 ] and spaceborne sensors [ 14 , 15 ], to name just a few of them. This phenomenon is also undesirable in the case of the use of computational imaging algorithms, such as the creation of high dynamic range (HDR) images [ 16 , 17 ], the stitching of static images to create panoramic [ 18 , 19 , 20 ] or mosaic images [ 3 , 4 , 5 , 6 , 21 ], as well as a panoramic real-time view [ 22 ]. Vignetting also affects the results of image analysis, including the results obtained using neural networks [ 23 , 24 ].…”

A Smooth Non-Iterative Local Polynomial (SNILP) Model of Image Vignetting

“…The effect of vignetting is undesirable in many applications, especially when there is a need for stitching images for creating panoramic images [3,4] or mosaic images [5,6], radiometric, or quantitative analysis of images [7,8], e.g., in such areas as microscopy [6,9,10], micro CT imaging [11], and remote sensing [12,13]. The best way to reduce vignetting is to remove its causes, e.g., by the usage of a lens with appropriate characteristics or by careful choice of exposure parameters.…”