Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution

Abstract

The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

Cryofixation during live‐imaging enables millisecond time‐correlated light and electron microscopy

“…In addition to correlating light and electron microscopy, the in situ cryofixation system permits the correlation of other imaging modalities in which cryogenic operation is advantageous. Cryoarresting the sample permits, for example, longer exposure times in fluorescence microscopy to increase photon collection without bleaching, phototoxic side effects, or artefacts from sample dynamics (Kaufmann et al, 2014;Masip et al, 2016). As many fluorophores emit orders of magnitude more photons at cryogenic temperature than at room temperature (Kaufmann et al, 2014;Li et al, 2015;Weisenburger et al, 2017), localisation microscopy can achieve significant resolution improvements under cryogenic conditions (Li et al, 2015;Weisenburger et al, 2017).…”

Artifact Formation in Single Molecule Localization Microscopy

“…For a systematic comparison see the publications of Sage et al [25] [26]. Within our discussion we will focus on QuickPALM [36], the first openly available SMLM software, ThunderSTORM [37], a tool that is very widespread in the SMLM community [38] [39] [40] and FIRESTORM. While QuickPALM uses a center-of-mass approach, ThunderSTORM offers a multitude of fitting and (post-) processing modalities.…”

Let There Be Light!

“…Another approach is to use low temperatures. By freezing the cell to around −40 °C for imaging and thereafter warming it up to 37 °C, it is possible to reduce photo-toxicity in the cell and use long exposure times [30]. …”