Yaolong Li, Bing Shi, Zhen‐Li Huang
ABSTRACT Scientific cameras are essential to modern fluorescence microscopy, serving as the “digital eye” that captures the foundational data for discovery. They play a particularly critical role in single‐molecule localization microscopy (SMLM), where camera performance directly determines the final image's resolution and fidelity. The technological evolution from highly sensitive EMCCDs to fast, large‐format sCMOS sensors has significantly expanded the frontiers of SMLM, enabling higher throughput and novel quantitative applications. While the maturation of these two technologies enables their complementary coexistence, it simultaneously introduces complex trade‐offs that pose significant challenges for technical beginners and biomedical researchers. In this perspective, we highlight these technological advancements. We examine the current challenges in camera selection and propose a comprehensive framework that spans the full workflow, from application‐oriented camera selection to system integration and data management. Finally, we outline the future perspectives for next‐generation detectors to ensure the continued advancement of SMLM as a premier tool in the life sciences.