Live cell imaging of heavy-ion-induced radiation responses by beamline microscopy

To study the dynamics of protein recruitment to DNA lesions, ion beams can be used to generate extremely localized DNA damage within restricted regions of the nuclei. This inhomogeneous spatial distribution of lesions can be visualized indirectly and rapidly in the form of radiation-induced foci using immunocytochemical detection or GFP-tagged DNA repair proteins. To analyze faster protein translocations and a possible contribution of radiation-induced chromatin movement in DNA damage recognition in live cells, we developed a remote-controlled system to obtain high-resolution fluorescence images of living cells during ion irradiation with a frame rate of the order of seconds. Using scratch replication labeling, only minor chromatin movement at sites of ion traversal was observed within the first few minutes of impact. Furthermore, time-lapse images of the GFP-coupled DNA repair protein aprataxin revealed accumulations within seconds at sites of ion hits, indicating a very fast recruitment to damaged sites. Repositioning of the irradiated cells after fixation allowed the comparison of live cell observation with immunocytochemical staining and retrospective etching of ion tracks. These results demonstrate that heavy-ion radiation-induced changes in sub-nuclear structures can be used to determine the kinetics of early protein recruitment in living cells and that the changes are not dependent on large-scale chromatin movement at short times postirradiation. © 2005 by Radiation Research Society.

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential

Reasons for performing study: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. Objectives: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. Methods: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T-2-weighted fast spin echo (FSE-T-2), and T-1-weighted gradient echo (GRE-T-1) sequences. Results: In T-2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. Conclusion: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (SIN) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased SIN. SIN can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. Potential relevance: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.

The discriminative bilateral filter: An enhanced denoising filter for electron microscopy data

Advances in three-dimensional (313) electron microscopy (EM) and image processing are providing considerable improvements in the resolution of subcellular volumes, macromolecular assemblies and individual proteins. However, the recovery of high-frequency information from biological samples is hindered by specimen sensitivity to beam damage. Low dose electron cryo-microscopy conditions afford reduced beam damage but typically yield images with reduced contrast and low signal-to-noise ratios (SNRs). Here, we describe the properties of a new discriminative bilateral (DBL) filter that is based upon the bilateral filter implementation of Jiang et al. (Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chin, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struc. Biol. 128, 82-97.). In contrast to the latter, the DBL filter can distinguish between object edges and high-frequency noise pixels through the use of an additional photometric exclusion function. As a result, high frequency noise pixels are smoothed, yet object edge detail is preserved. In the present study, we show that the DBL filter effectively reduces noise in low SNR single particle data as well as cellular tomograms of stained plastic sections. The properties of the DBL filter are discussed in terms of its usefulness for single particle analysis and for pre-processing cellular tomograms ahead of image segmentation. (c) 2006 Elsevier Inc. All rights reserved.