Expansion microscopy on Drosophila spermatocyte centrioles

Abstract

Drosophila spermatocyte centrioles are ideal for imaging studies. Their large, characteristic V conformation is both easy to identify and measure using standard imaging techniques. However, certain detailed features, such as their ninefold symmetry, are only visible below the diffraction limit of light. This is therefore a system that can benefit from the increased effective resolution potentially achievable by expansion microscopy. Here, I provide detailed protocols of two types of expansion microscopy methodologies applied to Drosophila spermatocyte centrioles, and discuss which is able to achieve the highest effective resolution in this system. I describe how to precisely measure these organelles post-expansion, and discuss how they can therefore be used as molecular rulers to troubleshoot and compare expansion techniques. I also provide protocols to combine expansion microscopy with super-resolution imaging in this tissue, discussing potential pitfalls. I conclude that expansion microscopy provides an effective alternative for thick tissues that are not amenable for traditional super-resolution techniques.