Rapid lightsheet fluorescence imaging of whole Drosophila brains at nanoscale resolution by potassium acrylate-based expansion microscopy

Xuejiao Tian, Tzu-Yang Lin, Po-Ting Lin, Min-Ju Tsai, Hsin Chen, Wen-Jie Chen, Chia-Ming Lee, Chiao-Hui Tu, Jui-Cheng Hsu, Tung-Han Hsieh, Yi-Chung Tung, Chien-Kai Wang, Suewei Lin, Li-An Chu, Fan-Gang Tseng, Yi-Ping Hsueh, Chi-Hon Lee, Peilin Chen and Bi-Chang Chen ()
Additional contact information
Xuejiao Tian: Academia Sinica
Tzu-Yang Lin: Academia Sinica
Po-Ting Lin: Academia Sinica
Min-Ju Tsai: Academia Sinica
Hsin Chen: Academia Sinica
Wen-Jie Chen: National Cheng Kung University and Academia Sinica
Chia-Ming Lee: Academia Sinica
Chiao-Hui Tu: Academia Sinica
Jui-Cheng Hsu: Academia Sinica
Tung-Han Hsieh: Academia Sinica
Yi-Chung Tung: Academia Sinica
Chien-Kai Wang: National Taiwan University
Suewei Lin: Academia Sinica
Li-An Chu: National Tsing Hua University
Fan-Gang Tseng: Academia Sinica
Yi-Ping Hsueh: Academia Sinica
Chi-Hon Lee: Academia Sinica
Peilin Chen: Academia Sinica
Bi-Chang Chen: Academia Sinica

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: Abstract Taking advantage of the good mechanical strength of expanded Drosophila brains and to tackle their relatively large size that can complicate imaging, we apply potassium (poly)acrylate-based hydrogels for expansion microscopy (ExM), resulting in a 40x plus increased resolution of transgenic fluorescent proteins preserved by glutaraldehyde fixation in the nervous system. Large-volume ExM is realized by using an axicon-based Bessel lightsheet microscope, featuring gentle multi-color fluorophore excitation and intrinsic optical sectioning capability, enabling visualization of Tm5a neurites and L3 lamina neurons with photoreceptors in the optic lobe. We also image nanometer-sized dopaminergic neurons across the same intact iteratively expanded Drosophila brain, enabling us to measure the 3D expansion ratio. Here we show that at a tile scanning speed of ~1 min/mm3 with 1012 pixels over 14 hours, we image the centimeter-sized fly brain at an effective resolution comparable to electron microscopy, allowing us to visualize mitochondria within presynaptic compartments and Bruchpilot (Brp) scaffold proteins distributed in the central complex, enabling robust analyses of neurobiological topics.

Date: 2024
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DOI: 10.1038/s41467-024-55305-8

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