Single molecule localization microscopy to study neuronal microtubule organization

The highly complex and polarized morphology of neurons is established by the cytoskeleton , a network of protein polymers , such as F-actin and microtubules , and associated proteins that provide shape and strength. In addition to providing structural support , microtubules serve as tracks for long-range active transport driven by dynein and kinesin motor proteins . To better understand how microtubule organization underlies the establishment and maintenance of neuronal architecture , better mapping of the neuronal microtubule network and its associated proteins is essential.
Different fluorescence microscopy techniques are commonly used to explore the organization of the microtubule cytoskeleton . The resolution of these techniques is, however, limited by diffraction to approximately 250 nm, which makes them not suitable for nanoscale mapping of microtubule properties. Super-resolution microscopy techniques that rely on single molecule localization (Single Molecule Localization Microscopy ; SMLM ) combine high protein specificity , multi-color imaging , and a resolution in the order of 5–50 nm, making it an ideal tool to study the neuronal cytoskeleton and its properties.
In this chapter, we discuss the theory behind SMLM , labeling strategies for the fluorescent probes , describe a workflow and a detailed protocol for fixation and immunostaining of neuronal microtubules , and provide some tips for successful super-resolution imaging , data analysis , and image reconstruction .