Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy

Abstract

SummaryFluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/ or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.

Optimal Multiresolution Blending of Confocal Microscope Images

“…Another class of methods matches the histograms of image stacks [28,29]. Other robust estimation methods have been proposed to remove outliers and adapt structures to compensate for the loss of intensity in microscope images [30].…”

Imaging of Fluorophores in Chromatographic Beads, Reconstruction of Radial Density Distributions and Characterisation of Protein Uptaking Processes

“…In our setting "reconstruction" has the following meaning: The true density of the fluorophores in a xy-plane perpendicular to the optical axis and of distance z from the microscopic lense is estimated using the data of the set of CLSM-slices which are closer to the lense. For a sound description of a robust incremental compensation of the light attenuation in 3D fluorescence microscopy applied to cellular structures is given by Kervrann et al (2004). In Susanto et al (2006a) the light attenuation effects in CLSM data of spherical beads are corrected in two steps.…”

“…In the present article we follow the approach of Kervrann et al (2004) and apply a gradient-based image correction. We start from the differential equation for the unknown light intensity describing the absorption along the path of the incoming and reflected light.…”

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

“…Another approach lies in the least squares optimization with brightness and contrast being optimized parameters exploiting all image pixel values (Kervrann et al, 2004;Periaswamy and Farid, 2003). These techniques are generally highly sensitive to noise present in images, which is practically always the case of CLSM images.…”