Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

Abstract

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ~65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ~30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 which localizes to disc microribbons, and the functionally unknown DAP16263 which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

X10 expansion microscopy enables 25‐nm resolution on conventional microscopes

“…X10 thus provides a toolset for cheap (< 2$ for all reagents used in one experiment, except antibodies) multi-color super-resolution imaging, which can be performed on widely available epifluorescence setups. This procedure provides a resolution that is superior to current combinations of conventional expansion microscopy and super-resolution, as published very recently using structured illumination [31], since this resulted in a resolution of~30 nm, lower than A B C Figure 3. The resolution of X10 is~25 nm.…”

Amplified Expansion Stimulated Emission Depletion Microscopy

“…In principle, super‐resolution instruments can be used with ExM samples to harness the advantages of both methods. Structured illumination microscopy (SIM), for example, was applied to expanded samples to achieve resolutions comparable with those of SMLM, whereas STED combined with ExM (ExSTED) produced a resolution of 10 nm . However, ExSTED cannot utilize the highest STED resolution because photobleaching prevents the use of the maximum depletion intensity.…”

Expansion microscopy

“…Will it be possible to achieve an even higher resolution by imaging an expanded specimen with super‐resolution microscopes? Until today, two studies have demonstrated such super super‐resolution imaging, one with photoactivated localization microscopy (Tillberg et al ., ) and the other one with SIM (Halpern et al ., ). In both the studies, super‐resolution microscopes were used to image a 4.5‐fold expanded biological specimens and a resolution higher than 60‐nm was achieved.…”

“…(E-H) Reproduced with permission from (Ku et al, 2016);Copyright 2016, Nature Publishing Group. ] with photoactivated localization microscopy (Tillberg et al, 2016) and the other one with SIM (Halpern et al, 2017). In both the studies, super-resolution microscopes were used to image a 4.5-fold expanded biological specimens and a res-olution higher than 60-nm was achieved.…”

“…Tel: 82-31-299-4403; e-mail: jbchang03@skku.edu a swellable hydrogel. The very first version of the expansionbased imaging techniques was demonstrated by Prof. Ed Boyden's group in 2015 (Chen et al, 2015) and multiple variants and applications of this technique have been published until today (Tillberg et al, 2016;Chozinski et al, 2016;Ku et al, 2016;Chen et al, 2016;Chang et al, 2017;Zhao et al, 2017;Halpern et al, 2017). In this review, we introduce the basic principle of expansion-based tissue imaging technique and its variants and their pros and cons.…”