TY - JOUR
T1 - Superresolution imaging of Drosophila tissues using expansion microscopy
AU - Jiang, Nan
AU - Kim, Hyeon Jin
AU - Chozinski, Tyler J.
AU - Azpurua, Jorge E.
AU - Eaton, Benjamin A.
AU - Vaughan, Joshua C.
AU - Parrish, Jay Z.
N1 - Funding Information:
This work was supported by a National Institutes of Health (NIH) grant (NINDS R01 NS076614), a UW Research Innovation award, and startup funds from the University of Washington (J.Z.P.); by an NIH grant (NIMH R01 MH115767), a Burroughs-Wellcome Career Award at the Scientific Interface, and startup funds from the University of Washington (J.C.V.); and by National Science Foundation Graduate Research Fellowship DGE-1256082 (T.J.C.). We thank the Center on Human Development and Disability for assistance with TEM sample preparation. Anti-perlecan antibodies were provided by Stefan Baumgartner, and the Brp antibody was obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development (NICHD) of the NIH and maintained at the University of Iowa. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.
PY - 2018/6/15
Y1 - 2018/6/15
N2 - The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.
AB - The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.
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U2 - 10.1091/mbc.E17-10-0583
DO - 10.1091/mbc.E17-10-0583
M3 - Article
C2 - 29688792
AN - SCOPUS:85048889621
VL - 29
SP - 1413
EP - 1421
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
SN - 1059-1524
IS - 12
ER -