Samples were transferred onto coverglass in a drop of PBS, moved to the desired position/orientation with forceps, and excess liquid was wicked away to ensure that samples adhered to the coverglass. epithelial ADRBK1 cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to tissues and underscores the importance of tissue-specific optimization of ExM procedures. INTRODUCTION Analysis of intercellular interactions and intracellular structures often requires optical resolution below the diffraction limit of light (250 nm). While several methods have been developed for superresolution imaging of biological samples using specialized microscopes (Huang tissues lacking a rigid cuticle are compatible with established ExM protocols, as has been also shown in two recent reports (Cahoon tissues, facilitating analysis of structural elements that cannot be accurately analyzed with standard optical microscopy, and we demonstrate the power of this approach in three experimental contexts. First, we show that ExM allows for high-resolution analysis of presynaptic active zone (AZ) structure at the larval neuromuscular junction (NMJ) and that analysis of these structures with standard confocal microscopy prospects to systematic sampling errors. Second, we identify age–dependent changes in adult AZ structure using ExM. Third, we analyzed cellCcell interactions in the larval peripheral nervous system using ExM and found that epithelial ensheathment of somatosensory dendrites is usually more prevalent than previously reported, underscoring the likely importance of this intercellular conversation. Altogether, these studies establish ExM as an accessible superresolution imaging platform amenable to analysis of diverse tissues. RESULTS AND Conversation Expansion of tissues with minimal distortion Prior studies demonstrated several specimens that are amenable to ExM (Chen development, we first examined whether ExM could be applied to intact embryos. To this end, we fixed embryos using a heptane/formaldehyde fixative and processed them for ExM, which includes gelation, digestion, and growth steps (Physique 1A). Using this approach, embryos were readily expanded 4 without obvious tearing or distortion (Physique 1B). To assess the fidelity of growth, we recorded images of embryos before and after growth. We then quantified distortion in the sizes by comparing postexpansion images to digitally expanded preexpansion images with a nonrigid deformation algorithm (Supplemental Figures S1 and S2). We found that lateral AB-680 distortion was generally below 1C2% over a range of length scales. Open in a separate window Physique 1: Isotropic growth of tissues for fluorescence microscopy. (A) ExM workflow. (BCD) Correlative pre- and postexpansion imaging of tissue. embryos (B), larval brains (C), and larval body walls (D) were stained with 4,6-diamidino-2-phenylindole (DAPI) and imaged both before and after growth. Preexpansion (inset) and postexpansion images are shown at the same magnification, such that postexpansion images are approximately four occasions larger than the corresponding preexpansion images. Next, we examined the compatibility of isolated tissue with ExM. Much like embryos, formaldehyde-fixed larval brains were readily expanded without gross distortion (Physique 1C). Staining larval brains with anti-Fas II antibody (Hummel sizes (Supplemental Physique S1), allowing us to generate strong steps of lateral and axial distortion. As in embryo preps, distortion in expanded larval brains was generally low ( 3%) over length scales of up to 30 m (Supplemental Physique S1), comparable to results reported for ExM of other tissue AB-680 samples (Chen tissues and that fine structural elements are preserved during growth of complex cellular assemblies, including whole-embryo preparations. Open in a separate window Physique 4: ExM analysis of age-related changes in AZ structure. Brp staining at the CM9 NMJ in unexpanded (A) and expanded (B, C) tissue of 10- or 65-d adult flies. In unexpanded samples, Brp immunoreactivity appeared as regularly shaped spherical structures (A) as well as larger structures possibly representing clusters of AZs (A). Arrowheads mark elongated puncta that likely represent clusters of joined AZs that cannot be resolved in unexpanded tissue (A) and clusters of joined AZs resolved by ExM (C). (D) Box plots depicting AZ area measurements using ExM. In this and subsequent panels, boxes mark 1st and 3rd quartiles, bands mark medians, whiskers mark 1.5 interquartile range, and outliers are shown AB-680 as open circles. **, 0.01 compared with 10-d adults, one-way ANOVA with AB-680 a post hoc Dunnetts test. (E) The.