Single-Molecule Localization Microscopy with Fixed Photoactivatable Fluorescent Proteins and Direct Stochastic...

Fluorescence microscopy has become one of the most widely used tools in modern cell biology. By selectively labelling molecules of interest with fluorescent tags, researchers can visualize the structural organization, distribution, and clustering of proteins within their native cellular environment. Single-molecule localization microscopy (SMLM) has extended fluorescence imaging by enabling spatial resolution far beyond the diffraction limit of light. Among SMLM techniques, fixed Photoactivated Localization Microscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM), and Direct Stochastic Optical Reconstruction Microscopy (dSTORM) provide access to subcellular organization below the diffraction limit that is otherwise difficult to achieve through conventional or even intermediate super-resolution methods, such as Structured Illumination Microscopy (SIM) or Stimulated Emission Depletion microscopy (STED). These techniques attain super-resolution by temporally separating fluorophore emission events, localizing individual molecules with high precision, and assembling their positions into a reconstructed image. Despite the recent advances of newer super-resolution modalities, fixed PALM and (d)STORM remain widely used, due to their molecular specificity, accessibility, and broad applicability across diverse biological systems. This chapter briefly introduces the core principles of SMLM, situates PALM and (d)STORM within the evolution of super-resolution microscopy, and provides detailed protocols for the image acquisition and data analysis of fixed-cell PALM and dSTORM imaging in monolayer cultures, such as PC12 cells and primary hippocampal neurons. The workflow described here is compatible with standard SMLM platforms and uses Zeiss ZEN Black 2012 and Abbelight NEO software for reconstruction and analysis.