Virtual Orientation Tools (VOTj): Fiji plugins for object centering and alignment

Abstract

Standardizing image datasets is essential for facilitating overall visual comparisons and enhancing compatibility with image-processing workflows. One way to achieve homogeneity for images containing a single object is to align the object to a common orientation. Here, we propose the Virtual Orientation Tools (VOTj): a set of Fiji plugins to center and align an object of interest in images to a vertical or horizontal orientation. To process an image, the plugin requires either a mask outlining the object or a rough annotation of the object directly drawn by the user in the image. The current object orientation is retrieved using Principal Component Analysis (PCA), from which the optimal alignment is derived. The plugins support multi-dimensional images to allow, e.g., aligning individual time points of a time-lapse. The tools can be used for a variety of samples and imaging modalities. Besides, the plugins enable the interactive alignment of a list of images from a directory for batch execution and can be included in custom image-processing workflows using macro-recording.

Figure 1. Virtual Orientation Tools (VOTj) for object alignment.

(A) Left: Overview of the VOTj plugin workflow and its application in aligning and centering a zebrafish embryo with the corresponding mask or user annotation. Center: User interface panel. Right: Resulting output images with and without the option of enlarging the canvas to prevent cropping original image regions. (B) An illustrative subset of zebrafish embryos imaged in a 96-well plate. Without dedicated mounting, these embryos adopt random orientations. (C) Corresponding images after transformation by the Virtual Orientation Tool plugin, using the option to enlarge the canvas. (D) Montage showing the centered and aligned zebrafish embryos across a full 96-well plate. The aligned images were cropped to display only the embryos. (E-F) The tool can be used to align various samples and is compatible with multi-dimensional images such as Z-stacks and time-lapse images, as shown in (E, F) for the alignment of a fluorescently labeled fly brain and a cell in mitosis, respectively. Input stack (left) and aligned stack (right). All images are background-corrected.