Ji-Seon Lim ¹, Hyun Jin Kim ², Ikbum Park ³, Sungwook Woo ¹, Joung-Hun Kim ^2,4*, Joon Won Park ^1,4*

¹Department of Chemistry, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Republic of Korea.
²Department of Life Sciences, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Republic of Korea.
³Analysis and Assessment Research Center, Research Institute of Industrial Science and Technology, 67 Cheongam-Ro, Nam-Gu, Pohang 37673, Republic of Korea.
⁴Institute of Convergence Science, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea.

^*Corresponding author.

Conventional methods for studying the spatial distribution and expression level of proteins within neurons have primarily relied on immunolabeling and/or signal amplification. Here, we present an atomic force microscopy (AFM)-based nanoscale force mapping method, where Anti-LIMK1-tethered AFM probes were used to visualize individual LIMK1 proteins in cultured neurons directly through force measurements. We observed that the number density of LIMK1 decreased in neuronal somas after the cells were depolarized. We also elucidated the spatial distribution of LIMK1 in single spine areas and found that the protein predominantly locates at heads of spines rather than dendritic shafts. The study demonstrates that our method enables unveiling of the abundance and spatial distribution of a protein of interest in neurons without signal amplification or labeling. We expected that this approach should facilitate the studies of protein expression phenomena in depth in a wide range of biological systems.