A Bi-channel Aided Stitching of Atomic Force Microscopy Images

Microscopy is an essential tool in scientific research, enabling the visualization of structures at micro- and nanoscale resolutions. However, the field of microscopy often encounters limitations in field-of-view (FOV), restricting the amount of sample that can be imaged in a single capture. To overcome this limitation, image stitching techniques have been developed to seamlessly merge multiple overlapping images into a single, high-resolution composite. The images collected from microscope need to be optimally stitched before accurate physical information can be extracted from post analysis. However, the existing stitching tools either struggle to stitch images together when the microscopy images are feature sparse or cannot address all the transformations of images. To address these issues, we propose a bi-channel aided feature-based image stitching method and demonstrate its use on AFM generated biofilm images. The topographical channel image of AFM data captures the morphological details of the sample, and a stitched topographical image is desired for researchers. We utilize the amplitude channel of AFM data to maximize the matching features and to estimate the position of the original topographical images and show that the proposed bi-channel aided stitching method outperforms the traditional stitching approach. Furthermore, we found that the differentiation of the topographical images along the x-axis provides similar feature information to the amplitude channel image, which generalizes our approach when the amplitude images are not available. Here we demonstrated the application on AFM, but similar approaches could be employed of optical microscopy with brightfield and fluorescence channels. We believe this proposed workflow will benefit the experimentalist to avoid erroneous analysis and discovery due to incorrect stitching.