Microtomy is a standard technique in biological and medical sample preparation. This technique is often used for 2D microscopy, but it can easily be extended to 3D analysis using AutoAligner.

AutoAligner reads a stack of digitally imaged serial sections and automatically aligns the single images, building up a 3D image that can be explored to Imaris. Each image is aligned in translation and rotation relative to its precursor image in the sequence. The transformations are reported for each individual section.

With AutoAligner you can also visually align images. In addition to a fully automated mode, AutoAligner provides manual control for neighboring images that are too different from each other for automatic alignment. The visual alignment tool uses fast display techniques for aligning images in real time. Simply click and rotate or click and translate the images as neighboring sections are superimposed over each other. The software also allows the operator to specify fiducially markers for alignment of the images.

AutoAligner works for both light and electron micrographs and thus opens the door to 3D microscopy! Since the resulting series of images from AutoAligner can be directly loaded into Imaris, most any data can be visualized and analyzed in 3D.

Features Include:

(1) Image Handling
AutoAligner produces 3D images from stacks of 2D images. It aligns serial sections produced in different formats.

• Microscopical Images – AutoAligner allows alignment of images form from light, confocal and electron microscopes.

• Mechanical Sections – AutoAligner allows alignment of a series of photomicrographs of cryo-, paraffin- or any other histological section.

• File Formats –Easily load Imaris files, TIFF series or virtually all formats used in confocal or optical microscopy.

(2) Pre-Processing
AutoAligner provides various pre-processing functions to prepare layers for alignment.

• Background Value - The appropriate choice of the background value is crucial for a good automatic alignment and should have a value that is similar to the mean intensity value along the borders of all images to be aligned. This allows alignment to be completed on structures rather than background.

• Normalization - Single layers/images can be acquired with different exposure times or have different thickness. Therefore some of the images differ visibly in the average intensity value. Normalization should be performed, to automatically calculate and apply an average contrast and brightness (linear normalization) to all layers, which helps with successful alignment.

• Inverting Intensity Values - It is of the utmost importance that the structures the automatic alignment has to be performed upon have the highest intensity values, i.e., are the brightest and therefore easiest to detect in the image. If these structures appear to be darker than the other areas of the image, the image can be inverted for the alignment process.

(3) Automatic Alignment
AutoAligner provides a tool for automatic alignment based on intensity values. Specific regions in the image can be selected and the calculation will only be performed on those regions.

• Selected Regions – Automatic alignment can be calculated on a user-defined region of the image layers to save computing time.

• Automatic Alignment Parameters – Basic parameters (such as Background Intensity, Type of Alignment, Choice of Channels) as well as advanced parameters (such as resampling factor, accuracy and restriction) for auto alignment can be set manually or automatically to achieve the best possible result.

• Resampling Factor - For a faster calculation the alignment can be calculated on a smaller, resampled version of the loaded data set.

• Choice Of Layers – Choose alignment of the whole stack calculated with the last chosen parameters or choose alignment of the currently visible layer with respect to the previous one for correction after an automatic alignment has been performed upon the entire data set.

(4) Correction Of An Image Layer Shift
AutoAligner allows correction of an image layer shift via both automatic and manual options.

• Translation - This function shifts the image if the image stack is horizontally or vertically shifted and removes constant offsets in x and y between neighboring slices in the resulting stack.

• Rotation - This function shifts the image to remove constant rotation between neighboring slices in the resulting stack.

(5) Manual Alignment
AutoAligner provides a tool to manually align images if more flexibility and visual control is needed for alignment because automatic alignment has produced some errors. This mode allows undo and reset of movements applied to single layers.

• Real-Time Interaction – AutoAligner allows you to shift and rotate image layers manually in real time.

• Fine-Tuning – Arrow heads and angels allow fine-tuning of the manual alignment to achieve the best possible accuracy.

• Overlay Modes – AutoAligner provides different overlay modes to visually guide aligning images. Choose between gray or colored overlays for this alignment.

• Quality Calculation - The quality of the manual alignment can be estimated by a Quality Update. AutoAligner computes an alignment quality factor for each slice pair, which is expressed as a function of the normalized cross-correlation coefficient, and can be color-coded as defined in the preferences for your convenience. This allows you to quickly assess the accuracy of each alignment.

(6) Post-Processing
Post-Processing actions are performed on the aligned data set before export. This provides a powerful tool for replacing missing layers or cropping the dataset so that it can be utilized for visualization.

• Interpolation – AutoAligner will replace a misaligned or bad looking layer with an artificial layer, which is interpolated from the adjacent layers.

• Cropping – The Crop function detects the common region of all overlaid layers. The cropping is performed automatically and the result is displayed in the main screen, and can now be easily be processed for further 3D volume rendering. This function is necessary to get rid of uncommon edges in the aligned image and to give better visualization results.

• 3D View – Dependent upon availability, Imaris can start automatically after the alignment is complete for further 3D image visualization and analysis.