Stitching of microscopic images is a technique used to combine multiple overlapping images (tiles) from biological samples with a limited field of view and high resolution to create a whole slide image. Image stitching involves two main steps: pairwise registration and global alignment. Most of the computational load and the accuracy of the stitching algorithm depend on the pairwise registration method. Therefore, choosing an efficient, accurate, robust, and fast pairwise registration method is crucial in the whole slide imaging technique. This paper presents a detailed comparative analysis of different pairwise registration techniques in terms of execution time and quality. These techniques included feature-based methods such as Harris, Shi-Thomasi, FAST, ORB, BRISK, SURF, SIFT, KAZE, MSER, and deep learning-based SuperPoint features. Additionally, region-based methods were analyzed, which were based on the normalized cross-correlation (NCC) and the combination of phase correlation and NCC. Investigations have been conducted on microscopy images from different modalities such as bright-field, phase-contrast, and fluorescence. The feature-based methods were highly robust to uneven illumination in tiles. Moreover, some features were found to be more accurate and faster than region-based methods, with the SURF features identified as the most effective technique. This study provides valuable insights into the selection of the most efficient and accurate pairwise registration method for creating whole slide images, which is essential for the advancement of computational pathology and biology.