Fig. 1: KA-ExM with Bessel lightsheet microscopy (∆BLX) for expansion and imaging of whole Drosophila brains.

a Synthesis of the in situ KA-ExM hydrogel. KPS potassium persulfate, TEMED tetramethylethylenediamine. b A free-standing KA hydrogel hosting an expanded fly brain stained with DAPI and illuminated by a 405 nm laser under a wide-field microscope. c The core ∆BLX system for imaging free-standing hydrogels comprises two ultrasonic piezo motors (U521) and one DC motor (M122) for sample translation, as well as a voice coil stage (V308) for sample scanning acquisition up to 7 mm. EO excitation objective, DO detection objective. Inset is a photograph of a centimeter-sized hydrogel in the water-filled imaging core. d Schematic for how a centimeter-sized gel is tiled by sample scanning using the voice coil motor. Note that there is no tiling in the z direction. e Maximum intensity projection (MIP) images of a series of expansions acquired by ∆BLX from an identical fly brain (grayscale image) with GFP-expressing dopaminergic neurons (heatmap) and nuclear staining (purple) first subjected to in situ KA-ExM and then iterative expansion (re-KA-ExM). Five independent gel expansions performed with similar results. f Enlarged 3D images for the ellipsoid body (EB) neuropil extracted from the expanded whole fly brain images in (e): heatmap images are the top (left) and side (right) views from an in situ KA-ExM brain and the two-color image (cyan and purple) is a top view of the re-KA-ExM sample. Note that the size of the original EB is shown in the yellow dashed box and all images have the same scale bar. Gamma correction applied to display colors on the 3D rendering.