Illumination Correction on Biomedical Images

RF-Inhomogeneity Correction (aka bias) artifact is an important research field in Magnetic Resonance Imaging (MRI). Bias corrupts MR images altering their illumination even though they are acquired with the most recent scanners. Homomorphic Unsharp Masking (HUM) is a filtering technique aimed at correcting illumination inhomogeneity, but it produces a halo around the edges as a side effect. In this paper a novel correction scheme based on HUM is proposed to correct the artifact mentioned above without introducing the halo. A wide experimentation has been performed on MR images. The method has been tuned and evaluated using the simulated Brainweb image database. In this framework, the approach has been compared successfully against the Guillemaud filter and the SPM2 method. Moreover, the method has been successfully applied on several real MR images of the brain (0.18 T, 1.5 T and 7 T). The description of the overall technique is reported along with the experimental results that show its effectiveness in different anatomical regions and its ability to compensate both underexposed and overexposed areas. Our approach is also effective on non-radiological images, like retinal ones

Non-common Path Aberration Correction in an Adaptive Optics Scanning Ophthalmoscope

The correction of non-common path aberrations (NCPAs) between the imaging and wavefront sensing channel in a confocal scanning adaptive optics ophthalmoscope is demonstrated. NCPA correction is achieved by maximizing an image sharpness metric while the confocal detection aperture is temporarily removed, effectively minimizing the monochromatic aberrations in the illumination path of the imaging channel. Comparison of NCPA estimated using zonal and modal orthogonal wavefront corrector bases provided wavefronts that differ by ~λ/20 in root-mean-squared (~λ/30 standard deviation). Sequential insertion of a cylindrical lens in the illumination and light collection paths of the imaging channel was used to compare image resolution after changing the wavefront correction to maximize image sharpness and intensity metrics. Finally, the NCPA correction was incorporated into the closed-loop adaptive optics control by biasing the wavefront sensor signals without reducing its bandwidth

High-resolution 3D optical microscopy inside the beating zebrafish heart using prospective optical gating

3D fluorescence imaging is a fundamental tool in the study of functional and developmental biology, but effective imaging is particularly difficult in moving structures such as the beating heart. We have developed a non-invasive real-time optical gating system that is able to exploit the periodic nature of the motion to acquire high resolution 3D images of the normally-beating zebrafish heart without any unnecessary exposure of the sample to harmful excitation light. In order for the image stack to be artefact-free, it is essential to use a synchronization source that is invariant as the sample is scanned in 3D. We therefore describe a scheme whereby fluorescence image slices are scanned through the sample while a brightfield camera sharing the same objective lens is maintained at a fixed focus, with correction of sample drift also included. This enables us to maintain, throughout an extended 3D volume, the same standard of synchronization we have previously demonstrated in and near a single 2D plane. Thus we are able image the complete beating zebrafish heart exactly as if the heart had been artificially stopped, but sidestepping this undesirable interference with the heart and instead allowing the heart to beat as normal

Multimodal wide-field two-photon excitation imaging: characterization of the technique for in vivo applications

We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications