Investigating pathogenic microorganisms is essential due to their significant impact on global health. However, working with them presents significant challenges to researchers. Their virulence requires strict biosafety measures, often necessitating to study model organisms instead. However, direct studies of the pathogens themselves remain crucial for advancing our understanding. Microscopy is a fundamental tool to study microorganisms. For live-cell microscopy, achieving high resolution while preserving cell viability, and ensuring biosafety compliance when studying pathogens are key considerations. In this work, I address these challenges by advancing high-resolution microscopy in specific areas, enabling time-lapse nano-characterization of pathogenic microorganisms in-vitro. Most commonly, optical microscopy is used for live-cell imaging of microorganisms. However, the diffraction of light restricts its resolution. Super-resolution techniques, such as structured-illumination microscopy, offer improved optical resolution unraveling fine details. Nonetheless, researchers often face limited access to super-resolution microscopes, forcing them to rely on lower-resolution alternatives. To promote wider accessibility to super-resolution techniques, we developed and released openSIM, an open-source add-on for structured illumination microscopy. The integrative add-on approach allows to improve the resolution of already existing fluorescence microscopes without the need to purchase a new instrument. Besides optical microscopy, high-resolution scanning probe microscopes like AFM enable nanometer-scale study of living biological samples, revealing both morphological and mechanical properties under physiological conditions. However, typical AFM sample holders do not fulfill biosafety standards because they do not present a closed system. I developed a hermetically sealed AFM sample chamber that fulfills biosafety compliance while maintaining the mechanical and optical compatibility necessary for correlated optical microscopy and AFM, thereby facilitating time-lapse nano-characterization of pathogenic microorganisms in-vitro. Finally, I investigated the effect of cell cleavage on growth and DNA replication in mycobacteria using correlated optical and AFM imaging combined with nano-manipulation to better understand mycobacterial cell cycle coordination. For this, I studied a M. smegmatis strain that forms cell chains due to impaired cell cleavage. External AFM-induced cell cleavage facilitated investigating the impact of cell cleavage on different cellular processes. Wag31, an essential protein of the elongation machinery, localizes to septa within chained cells and accumulates over time despite the lack of cell cleavage, but to a lower level than that of growing poles in normally dividing cells. AFM-induced cleavage led to growth without elevated Wag31 levels, suggesting that cell cleavage does not affect growth onset by influencing the Wag31 concentration. Additionally, cell cleavage influences DNA replication dynamics but is not essential for its initiation. These findings provide new insights into the coordination of cell growth, division, and DNA replication in mycobacteria. Together, this work demonstrates how advancements in high-resolution microscopy can overcome limitations in studying microorganisms, improving both accessibility to super-resolution microscopy and enabling nano-characterization of pathogenic microorganisms.