Challenges of Using Expansion Microscopy for Super‐resolved Imaging of Cellular Organelles

Abstract

Expansion microscopy (ExM) has been successfully used to improve the spatial resolution when imaging tissues by optical microscopy. In ExM, proteins of a fixed sample are crosslinked to a swellable acrylamide gel, which expands when incubated in water. Therefore, ExM allows enlarged subcellular structures to be resolved that would otherwise be hidden to standard confocal microscopy. Herein, we aim to validate ExM for the study of peroxisomes, mitochondria, nuclei and the plasma membrane. Upon comparison of the expansion factors of these cellular compartments in HEK293 cells within the same gel, we found significant differences, of a factor of above 2, in expansion factors. For peroxisomes, the expansion factor differed even between peroxisomal membrane and matrix marker; this underlines the need for a thorough validation of expansion factors of this powerful technique. We further give an overview of possible quantification methods for the determination of expansion factors of intracellular organelles, and we highlight some potentials and challenges.

Prelabeling Expansion Single‐Molecule Localization Microscopy with Minimal Linkage Error

“…Although the post-expansion intensity of Alexa 647 was significantly reduced by physical expansion and fluorophore loss [11] and the co-expressed mCherry-H2B was better retained after expansion, we used vimentin structures for measuring expansion factors since different cellular compartments expands with different expansion factors. [13] From the pre-and post-expansion images of the same fields of view (Figures 3 and S2), the average expansion factor was measured as 3.4 � 0.4 (mean � sd), consistent with the value obtained from gel diameters (Figure 1E) and previous works. [7a] To tackle the issue of label loss, we made linkable streptavidin by using the succinimidyl ester of 6-((acryloyl) amino)hexanoic acid (acryloyl-X SE; abbreviated AcX).…”

“…After ExM treatment, most labeled cells remained fluorescent and preserved characteristic vimentin structures (Figures 3B and S3 A). Although the post‐expansion intensity of Alexa 647 was significantly reduced by physical expansion and fluorophore loss [11] and the co‐expressed mCherry‐H2B was better retained after expansion, we used vimentin structures for measuring expansion factors since different cellular compartments expands with different expansion factors [13] . From the pre‐ and post‐expansion images of the same fields of view (Figures 3 and S2), the average expansion factor was measured as 3.4±0.4 (mean±sd), consistent with the value obtained from gel diameters (Figure 1E) and previous works [7a] …”

Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

“…Our data, however, also indicated that the degree of expansion was not identical for the studied cytoskeletal structures; while the axoneme expanded 4.8 fold, similar to the 4.7 expansion factor of the gel, the distance between the corset microtubules increased 6.0 fold. Leaving aside the potential caveats associated with estimating dimensions from EM data (Shah et al, 2015), which were used to calculate these expansion factors, similar and higher discrepancies in expansion factors of individual organelles were recently observed in various experimental systems and using a variety of ExM protocols (Büttner et al, 2020;Kubalová et al, 2020;Pernal et al, 2020;Pesce et al, 2019). Hence, it seems likely that based on their composition individual organelles and structures undergo differential expansion, precluding a straightforward interpretation of measured distances between entities of expanded specimens.…”

Imaging Nanoscale Nuclear Structures with Expansion Microscopy

“…The sample is digested with proteases and the addition of water results in volumetric expansion of the gel, with the aim of retaining the relative spatial organisation of the labels 9,10,17 . However, the presence of genomic DNA in the gel has been suggested to introduce distortions when expanding the nucleus, adversely affecting isotropic expansion 12,14,18 .…”

“…Expansion Microscopy (ExM) has the potential to overcome some of the problems posed by other SRM modalities. However, whilst ExM has been successfully applied to the analysis of cytoplasmic structures, concerns over differential nuclear expansion and controversy on how samples should be prepared for its investigation has limited ExM investigation of nanoscale structures in the nucleus [9][10][11][12][13][14] .…”