A square electron beam improves tiling in transmission electron microscopy (TEM), enabling high-resolution imaging with minimal exposure loss. Traditional TEMs use round beams, leading to imperfect tiling and uneven exposure, especially for dose-sensitive samples. This study introduces a square beam, which can be retrofitted into existing microscopes, allowing nearly perfect tiling and efficient data collection. The square beam is created using a C2 aperture with a square hole, enabling precise alignment with the sensor. This setup allows for minimal overlap in tiling, which is crucial for in situ tomography and single-particle analysis. The square beam also enhances throughput, allowing more targets to be imaged per stage movement compared to round beams. The study demonstrates that the square beam maintains high-resolution imaging and reduces exposure damage to the sample. The beam's square profile also allows for better alignment with the sensor, reducing stitching lines in montage tomography. The square beam was tested on various samples, including apoferritin and yeast lamellae, showing improved imaging quality and data collection efficiency. The study also highlights the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam offers a solution to the limitations of round beams in TEM, providing a more efficient and effective method for high-resolution imaging and tomography. The results show that the square beam can be used for cryo-electron microscopy and cryo-electron tomography, with minimal loss of resolution and improved data collection efficiency. The study also discusses the potential for using other non-circular beam shapes, such as rectangles and hexagons, for optimal tiling. The square beam is a significant advancement in TEM technology, offering improved imaging quality and data collection efficiency for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a promising solution for improving the efficiency and quality of TEM imaging, particularly for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a significant advancement in TEM technology, offering improved imaging quality and data collection efficiency for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a promising solution for improving the efficiency andA square electron beam improves tiling in transmission electron microscopy (TEM), enabling high-resolution imaging with minimal exposure loss. Traditional TEMs use round beams, leading to imperfect tiling and uneven exposure, especially for dose-sensitive samples. This study introduces a square beam, which can be retrofitted into existing microscopes, allowing nearly perfect tiling and efficient data collection. The square beam is created using a C2 aperture with a square hole, enabling precise alignment with the sensor. This setup allows for minimal overlap in tiling, which is crucial for in situ tomography and single-particle analysis. The square beam also enhances throughput, allowing more targets to be imaged per stage movement compared to round beams. The study demonstrates that the square beam maintains high-resolution imaging and reduces exposure damage to the sample. The beam's square profile also allows for better alignment with the sensor, reducing stitching lines in montage tomography. The square beam was tested on various samples, including apoferritin and yeast lamellae, showing improved imaging quality and data collection efficiency. The study also highlights the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam offers a solution to the limitations of round beams in TEM, providing a more efficient and effective method for high-resolution imaging and tomography. The results show that the square beam can be used for cryo-electron microscopy and cryo-electron tomography, with minimal loss of resolution and improved data collection efficiency. The study also discusses the potential for using other non-circular beam shapes, such as rectangles and hexagons, for optimal tiling. The square beam is a significant advancement in TEM technology, offering improved imaging quality and data collection efficiency for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a promising solution for improving the efficiency and quality of TEM imaging, particularly for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a significant advancement in TEM technology, offering improved imaging quality and data collection efficiency for dose-sensitive samples. The study provides a detailed methodology for creating and using the square beam, along with results from various experiments and data analysis. The results demonstrate the effectiveness of the square beam in improving tiling and data collection in TEM. The study also discusses the importance of calibration and adjustment of the P2 lens to achieve optimal imaging. The square beam is a promising solution for improving the efficiency and