Image registration aligns the common features of two images. The open-source Insight Toolkit (ITK, funded by the National Library of Medicine) provides a library of pluggable registration components, providing different transform types, image metrics, interpolators, and optimizers. Image registration produces a mathematical transformation that describes how one image has moved in relation to another. Applying registration methods to a video sequence allows one to analyze the motion of image elements. The collection of transformations obtained through this method will provide a way to create visual representations of the velocity flow present in mitosis videos. Registration of mitosis videos at a coarse scale will stabilize the video with respect to the spindle by removing the total motion of the spindle. We allow for rigid body transformations (rotations and translations), and use a mutual information image metric that accounts for varying intensities from image to image. Our goal is to create an interactive application that uses this texture- based tracking to examine the average motion of smaller scale features as well. Image noise and the bleaching of individual fluorophores makes spot-based tracking an unlikely accurate motion analysis method. Following small texture patches should make this video-based method more robust—by concentrating on smaller regions of the images, registration will provide a view of how kinetochores and microtubule regions move over time. Above: Schematic representation of image registration. Points on one image are mapped to points on another image through a transformation. Here, information about the total motion of the cell spindle over multiple frames is collected. Researchers believe that the dynamics of the mitotic spindle is key to understanding the healthy division of cells. Problems encountered during mitosis can lead to birth defects and some forms of cancer. The following components play a part in successful mitosis: Kinetochores, specialized protein complexes, bind to sites on the chromatids Kinetochore microtubules bind to the kinetochores and align the chromatids at the mitotic plate The composition of individual microtubules constantly changes—tubulin polymerizes at the kinetochore and de-polymerizes at the centrosomes Videos of cellular mitosis display this multi-scaled motion. The entire spindle moves from side to side and rotates. The microtubules are in motion independent of the cell while their composition changes. Sister kinetochores align at the mitotic plate while changing position relative to one another. Simultaneously quantifying these motions can be difficult. Video Analysis of Cellular Mitosis CISMM: Computer Integrated Systems for Microscopy and Manipulation Collaborators: Dr. Lisa Cameron, Professor Edward D. Salmon, UNC Department of Biology Project Lead: Russell Taylor Investigators: Brian Eastwood, Steven Pizer December 2003 The problem: Analyzing motion during cellular mitosis Our approach: Image registration at multiple scales Key scientific questions: How are sister kinetochores moving with respect to the spindle and in relation to each other? What is the motion of microtubules, independent of the total cell motion? Is there a way to examine the growth of microtubules at the kinetochores and their disassembly at the poles? Fixed ImageMoving Image Image Metric Optimizer Interpolator Transform Two images are registered using ITK components. A transform defines the legal range of motions. An optimizer determines the next set of transform parameters. An image metric determines how closely the two images are aligned. An interpolator calculates image values of the moving image at non- pixel points. Left: Diagram of components of mitosis (prometaphase). (Adapted from Campbell et al Biology. Pearson Education, Inc.) Right: Mitosis captured by a confocal microscope. Tubulin, the building block of microtubules, has been labeled with red fluorescing dye; kinetochores have been labeled green. Left: Key frames from a video of cellular mitosis. The spindle changes its orientation throughout. More subtle changes in relative kinetochore position are also present. Changes in the composition of individual microtubules are quite difficult to detect. Below: Key frames hand-aligned with respect to the spindle. The dark regions in the corners of the images indicate the difference between the fixed image (first frame) and each moving image.