title: 3D multicolor super-resolution imaging offers improved accuracy in neuron tracing.
abstract: The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America. melike.lakadamyali@icfo.es
authors.forename: Melike
authors.initials: M
authors.lastname: Lakadamyali
authors.forename: Hazen
authors.initials: H
authors.lastname: Babcock
authors.forename: Mark
authors.initials: M
authors.lastname: Bates
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhuang
authors.forename: Jeff
authors.initials: J
authors.lastname: Lichtman
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D003116:Color; D005260:Female; D006624:Hippocampus / Q000166:cytology / Q000648:ultrastructure; D007089:Image Enhancement / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D056347:Neuroanatomical Tract-Tracing Techniques / Q000379:methods*; D009474:Neurons / Q000166:cytology* / Q000648:ultrastructure; D011247:Pregnancy; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity