Recent Advances in Single‐Molecule Localization Based Super‐Resolution Imaging

Over the past two decades, single-molecule localization microscopy (SMLM) has developed into one of the most versatile and powerful tools for imaging biological structures and activities beyond the diffraction limit. Advances in techniques and probes enable the direct visualization of fine structures under sophisticated conditions, leading the research brighter and crisper at the single-molecule level. In this review, a brief introduction to the basic principle of SMLM is provided, as well as the core implementation processes for single-molecule localization. Then, the developed corelative methods of SMLM with other beneficial approaches in the recent 5 years are reviewed subsequently, highlighting the modifications for increased resolution, penetration depth, speed, and throughput. Next, the improvements in the fluorescent labeling probes are discussed together with the spectroscopic and chemical demands for the fluorophores. Cutting-edge achievements of SMLM in biological systems are summarized, focusing on how single-molecule localization imaging helps to clarify intracellular structures and processes that are relevant to cells, neurons, tissues, microorganisms, and pathology. Finally, the challenges as well as future trends in this field are demonstrated.