Super-resolution microscopy facilitates the visualization of cellular structures at a resolution approaching the molecular level. Especially, super-resolution techniques based on the localization of single molecules have relatively modest instrument requirements and are thus good candidates for adoption in bioimaging. However, their low-throughput nature hampers their applicability in biomolecular research and screening. Here, we propose a workflow for more efficient data collection, starting with the scanning of large areas using fast fluctuation-based imaging, followed by single-molecule localization microscopy of selected cells. To achieve this workflow, we exploit the versatility of DNA oligo hybridization kinetics with DNA-PAINT probes to tailor the fluorescent blinking toward high-throughput and high-resolution imaging. Additionally, we employ super-resolution optical fluctuation imaging (SOFI) to analyze statistical fluctuations in the DNA-PAINT binding kinetics, thereby tolerating much denser blinking and facilitating accelerated imaging speeds. Thus, we demonstrate 30–300-fold faster imaging of different cellular structures compared to conventional DNA-PAINT imaging, albeit at a lower resolution. Notably, by tuning the image medium and data processing though, we can flexibly switch between high-throughput SOFI (scanning an FOV of 0.65 mm × 0.52 mm within 4 min of total acquisition time) and super-resolution DNA-PAINT microscopy and thereby demonstrate that combining DNA-PAINT and SOFI enables one to adapt image resolution and acquisition time based on the imaging needs. We envision this approach to be especially powerful when combined with multiplexing and 3D imaging.

Image quality in single-molecule localization microscopy depends largely on the accuracy and precision of the localizations. While under ideal imaging conditions, the theoretically obtainable precision and accuracy are achieved; in practice, this changes if (field-dependent) aberrations are present. Currently, there is no simple way to measure and incorporate these aberrations into the point-spread function (PSF) fitting; therefore, the aberrations are often taken as constant or neglected altogether. Here we introduce a model-based approach to estimate the field-dependent aberration directly from single-molecule data without a calibration step. This is made possible by using nodal aberration theory to incorporate the field dependency of aberrations into our fully vectorial PSF model. This results in a limited set of aberration fit parameters that can be extracted from the raw frames without a bead calibration measurement, also in retrospect. The software implementation is computationally efficient, enabling the fitting of a full 2D or 3D dataset within a few minutes. We demonstrate our method on 2D and 3D localization data of microtubuli, nuclear pore complexes, and nuclear lamina over fields of view of up to 180 µm and compare it with Gaussian fitting, spline-based fitting, and a deep-learning-based approach.

As current challenges become more complex, bringing together people from different backgrounds to solve multifaceted problems has become crucial in the STEM field. There is growing recognition of the need to explicitly teach students the skills necessary to conduct inter- and transdisciplinary science, including communication, collaboration, reflection, and understanding of the research process. This set of skills is complex and requires acquiring knowledge and practice and changing attitudes. Moreover, these processes also require the cultivation of emotional skills, which are often neglected in the STEM field, especially in project-based learning programs that primarily emphasize technical expertise. As educators, we recognize our need to develop these competencies as well. This case study reports on the co-creation journey of an education program designed to teach collaboration with a strong emotional intelligence component in biomedical research to bachelor’s students while providing them with a project to practice. This program also provides an environment for scientists, PhD students, and us as an education design team to improve our skills. We report on what we have done and learned from each other and our students in 360º education, including the program design process evaluation, which tends to be overlooked in the development process of such educational programs.