Cryo-electron microscopy (cryo-EM) is a powerful technique extensively uti- lized for studying biological systems. It allows for high-resolution structural de- termination of macromolecules and visualization of cellular structures in their near-native environment.This thesis starts with a concise overview of the fundamental principles and applications of selective cryo-EM techniques with conventional transmission electron microscopy (TEM), such as single particle analysis (cryo-SPA), electron tomography (cryo-ET), and focused-ion-beam milling (cryo-FIB). Subsequently, applications of scanning-TEM (STEM) in vitrified hydrated biological systems are introduced, along with the progress made in four-dimensional STEM (4D- STEM) techniques. The challenges encountered in imaging thick biological specimens using conventional TEM techniques are highlighted, along with the limited applica- bility of conventional STEM for high-resolution structural determination due to collection efficiency limitations. To address these challenges, a 4D-STEM technique, tilt-corrected bright-field STEM (tcBF-STEM) is introduced, aiming to provide advantages for imaging thick dose-sensitive specimens by optimiz- ing the collection efficiency of coherent BF-STEM images. The implementation of tcBF-STEM with a pixelated detector optimized for STEM is demonstrated, along with the extraction of probe aberration information using tcBF imaging. Notably, tcBF-STEM enables the retrieval of information beyond the real-space scanning Nyquist limit, effectively mitigating the effects of specimen motion in cryo-EM experiments with a single tilt direction. The results of applying tcBF- STEM to imaging vitrified biological specimens, including vitrified apoferritin protein and E. coli bacteria, are presented. The potential of tcBF-STEM and its limitations are discussed at the end.