Controlled Grafting Expansion Microscopy

Abstract

Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.

Trifunctional Linkers Enable Improved Visualization of Actin by Expansion Microscopy

“…For example, by staining cells with AcX (0.1 mg/mL in PBS) prior to gelation, signal retention increased from 0.024 to 0.127 (5-fold) and from 0.046 to 0.199 (4-fold) for compounds 2 and 6 , respectively (Figure c). We rationalized that AcX signal retention improvement is due to the increased actin surface-bound acryloyl concentration enabling more efficient grafting of both actin filaments and TRITON molecules. , This finding is also consistent with the very recent paper of Ria and co-workers . Furthermore, we noticed a further improvement of signal retention of compound 6 from 0.199 to 0.243 when increasing the AcX concentration from 0.1 to 0.5 mg/mL (Figure c).…”

“…38,39 This finding is also consistent with the very recent paper of Ria and co-workers. 40 Furthermore, we noticed a further improvement of signal retention of compound 6 from 0.199 to 0.243 when increasing the AcX concentration from 0.1 to 0.5 mg/mL (Figure 3c). Inspired by these results, we explored the impact of crosslinking agents on signal retention of actin filaments in more detail.…”

“…However, we did not observe a significant improvement of signal retention at higher AcX concentrations. It has also been demonstrated that higher AcX concentrations lower the expansion factor that can be achieved, due to overgrafting of samples. , In addition, as aforementioned, by applying the antifluorophore antibody strategy, actin filaments can be visualized postexpansion with a similar brightness as with our optimized TRITON system. It is worth mentioning that in this method, the retained fluorescence signal originates from sufficient grafting of the fluorescently labeled antibodies, not phalloidin labels, and the majority of phalloidin conjugates is washed out postexpansion.…”

Differential labelling of human sub-cellular compartments with fluorescent dye esters and expansion microscopy

“…Availability of amines is likely to vary in relation to the anchoring step, given that AcX reacts with amines, 27 and also the effects of proteinase K hydrolysing peptide bonds, potentially exposing new amine groups in previously inaccessible regions, or losing protein fragments from the gel altogether. 28 Our investigation of labelling timepoints made use of the hydrophobic NHS ATTO647N, however it is possible that more hydrophilic esters may experience different time-specific staining pattern alterations. In addition, it is well documented that certain dyes are less resistant to bleaching during the expansion process.…”

Bio-orthogonal Glycan Imaging of Culture Cells and Whole AnimalC. eleganswith Expansion Microscopy

“…To enhance fluorescence and probe retention, multifunctional chemical linkers that directly graft fluorophores to ExM gels have recently been demonstrated for coupling various biomolecules including protein tags 31,32 , lipids 33,34 , and actin 35 . These approaches, parallelly termed trivalent anchoring (TRITON) 33 and label-retention expansion microscopy (LR-ExM) 31 , are attractive candidates for probing macromolecules in multicellular systems due to the small size of the reagents.…”