A precise light attenuation correction for confocal scanning microscopy with O(n^4/3) computing time and O(n) memory requirements for n voxels

Abstract

Some dyes and tissues observed by confocal fluorescence microscopy show remarkable opacity, caused by absorption and scattering within the scanned volume. The efficiency of the excitation and fluorescence process may vary by a factor of 10 from top to bottom regions even in not very extended scan volumes. Based on known attenuation properties of a specimen, the amount of damping is computed by integrating the attenuation along all light paths within the numerical aperture of the objective, Thus, to correct a single volume element (voxel), one has to take into account the damping within the whole extended conical volume between the lens and the focus, resulting in intolerable execution times. Common approaches reduce resolution or simplify the integration paths, thereby resulting in either a loss of fine resolution or showing a low resolution versus computation time ratio. This paper presents a more efficient reformulation of this spatial integration process without simplifying the physical background. This algorithm requires computing times slightly longer than those of programs using simplified physical and statistical approaches. However, the algorithm may be tuned to achieve a precision and stability comparable to exhaustive integration.

A hybrid 3D watershed algorithm incorporating gradient cues and object models for automatic segmentation of nuclei in confocal image stacks

“…Several sophisticated methods have been used to correct for depth-dependent signal attenuation (61,62). Unfortunately, the computational needs of these methods are high enough to preclude routine use on a large scale.…”

Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads

“…There have been a number of computational approaches for correcting confocal microscopy images for absorption and scattering (41)(42)(43)(44). Furthermore, mismatch effects between the movement of the focal plane and that of the specimen stage due to refractive index differences have been described previously (45)(46)(47).…”

Confocal laser scanning microscopy as an analytical tool in chromatographic research

“…Mathematical approaches to solve the attenuation problem apply physical models of the excitation and fluorescence emission phenomena [23,24,28,31,68,69]. In this approach, unknown model parameters (i.e., image correction parameters) are determined experimentally for the respective experimental systems without a change in system parameters.…”