Vignetting and photo-bleaching correction in automated fluorescence microscopy from an array of overlapping images

Abstract

We propose a novel acquisition scheme and non-parametric multi-image based method for correcting illumination in fluorescence images. Our approach measures changes in intensity of the subject by moving the microscope stage at regularly spaced intervals, and exploits this information to learn the correction function. The acquisition process and learning are performed prior to imaging, and take only a few minutes. Afterwards, images can be corrected for vignetting and photo-bleaching effects on the fly. Our approach can be implemented in any microscope with a motorized stage, and does not require a reference calibration slide. Experiments demonstrate that our method outperforms standard approaches to illumination correction. VIGNETTING AND PHOTO-BLEACHING CORRECTION IN AUTOMATED FLUORESCENCE MICROSCOPY FROM AN ARRAY OF OVERLAPPING IMAGES ABSTRACTWe propose a novel acquisition scheme and non-parametric multi-image based method for correcting illumination in fluorescence images. Our approach measures changes in intensity of the subject by moving the microscope stage at regularly spaced intervals, and exploits this information to learn the correction function. The acquisition process and learning are performed prior to imaging, and take only a few minutes. Afterwards, images can be corrected for vignetting and photobleaching effects on the fly. Our approach can be implemented in any microscope with a motorized stage, and does not require a reference calibration slide. Experiments demonstrate that our method outperforms standard approaches to illumination correction.

Improving reliability of live/dead cell counting through automated image mosaicing

“…Many methods have been proposed in the literature to attenuate vignetting [78]. Some of them rely on acquisition of reference images from an empty field [79], while others use directly the acquired images themselves to estimate the vignetting curvature [80]. Nevertheless, so far a general purpose solution for correcting the vignetting does not exist.…”

Shading Correction for Whole Slide Image Using Low Rank and Sparse Decomposition

“…right). Besides visual inspecti correction algorithm is further assessed using root-me e overlap regions, a popular criteria to measure the mos croscopy [1]. As reported in Table 1 Fig.…”

“…However, a common artifact that encumbers the use of automatic image processing techniques is intensity inhomogeneity present in microscopic images, also known as shading [1]. Intensity inhomogeneity may originate from non-uniform illumination, uneven sample thickness or camera nonlinearity.…”

µ-Mosaicing and Thermo-Mosaicing using Image In-painting

“…In literature [4,5,6], methods to remove the effects of vignetting from the multiple images acquired from a microscope has been presented. A general purpose and non-parametric method is presented in literature [4] to estimate the illumination field from the background while obtaining images by moving microscope holder.…”

“…However, the method is not suitable for specimen with very small background in the image. A non-parametric, multiimage based correction method and acquisition scheme for correcting the effects of vignetting and photo-bleaching in fluorescence microscopy has been implemented in [5]. The algorithm learns the correction function by automatically moving the microscope stage at regular intervals and by measuring the changes in intensity.…”