Cellular cartography: mapping the neuronal microtubule network using super-resolution microscopy

Abstract

Described in this thesis are the development and use of novel single molecule localization microscopy technologies to gain new insights into (neuronal) microtubule organization. The image quality of single molecule localization microscopy (SMLM) depends on a sound optical setup. Aberrations introduced by optical components result in optical distortions, lower photon counts, and a decreased localization precision. Avoiding aberrations or correcting them is therefore of great importance. We show that SMLM deep in biological samples is aided by the use of a deformable mirror to decrease aberrations. At the same time the deformable mirror can be used to encode information about the z-position of molecules allowing them to be localized with high accuracy in three dimensions. Optimized fixation protocols and fluorescent probes are just as important for high quality images. The immunostaining protocol described in this thesis allows SMLM of microtubules and microtubule associated proteins with low background signal and high labeling density. This protocol was used to show that microtubule organization drastically changes during neuronal development, and that the microtubule associated protein CAMSAP2 decorates MT minus-ends in neurons. Optimization of fluorescent probes in terms of brightness, return percentage, and size also leads to higher quality SMLM imaging. The commonly used bright monomeric red fluorescent protein mCherry turned out to be a high quality SMLM probe when exploiting a purely chemical caging mechanism with a return percentage up to 80%. Other novel probes very small in size, called tubulin nanobodies, were shown to enable investigation of individual microtubules in dense bundles, which is not possible with conventional antibodies. A property of microtubules that cannot be determined with immunostaining is their orientation. We showed, using a processive kinesin motor protein as a probe for SMLM. that the orientation of MTs can be determined in fixed cells and, surprisingly, that there is local orientational order in the dendritic MT array. In conclusion, described in this thesis are the development of novel SMLM methods and their use to find new insights in the neuronal microtubule organization.