Cyclic Expansion Microscopy: Expanding Biological Tissue through Multiple Cycles for Ultrastructure Imaging

Abstract

Expansion microscopy allows super resolution imaging of cellular structures by fluorescence microscopy. However, current protocols achieving large expansion factors (near 8 and beyond), are only applicable to cultured cells and thin tissue slices, but not to bulk tissue in general. Here, we present a method that allows unlimited cycles of expansion of bulk tissue with high isotropy, which we term as Cyc-ExM. The protocol uses identical gel recipe and denaturation reagents in each expansion cycle, which provides ease to the procedure. Cyc-ExM expands biological tissues ~9 fold in each dimension from 7 cycles of expansion and 17-27 fold from 10 cycles. The covalent staining procedure was also optimized to reveal ultrastructural features. Besides, Cyc-ExM is compatible with antibody labeling and multi-scale 3D imaging. Despite volume dilution of signal during expansion, we developed a signal amplification method achieving 8-fold increase in signal, which greatly facilitates the resolution of sub-organelle structures within various tissues.

Expanding the toolset of fluorescent covalent staining of biological samples by labeling carboxylate and phosphate groups

“…The tissue blocks were then processed for expansion microscopy using our in-house protocol (Cyclic Expansion Microscopy, Cyc-ExM) developed recently [9]. Briefly, for each cycle of expansion, the tissue was soaked in the gel monomer solution containing 3% (w/v) PFA, 20% (w/v) AA, and 0.1% (w/v) V50 (S15042-25 g, Yuanye Bio-Technology) for 4 h at 4 C and polymerized at 80 C for 2 h. The sample was then transferred to the denaturation solution (200 mM sodium dodecyl sulfate [SDS], 200 mM NaCl, 50 mM Tris-HCl, pH 9.0) and incubated at 80 C for 2 Â n h for the n-th cycle of expansion.…”

“…In order to minimize such irreversible crosslinks, which could resist expansion of the tissue during expansion microscopy, the amine stain was applied to the tissue before the carboxylate and phosphate stain. Next, we used our recently developed expansion protocol, Cyc-ExM [9], which has the advantage of low distortion induced by expansion. After six cycles of expansion, linear expansion factors of 3.66 ± 0.44, 4.71 ± 0.61, 3.65 ± 0.60, and 4.94 ± 0.65 were achieved for rat heart, liver, intestine, and kidney tissues, respectively.…”