Cryo-EM sample preparation using microfluidic cryofixation and cryo-FIB for time-resolved studies

Understanding the functions of biomolecules requires dynamic studies of their molecular structures. These critical conformational changes occur in a short time scale which makes it challenging to directly study the dynamics using conventional cryo-EM sample preparation, such as blotting. To overcome this limit, time-resolved cryo-EM techniques are developed, which utilize spray deposition of small droplets. The reactions can be initiated in a microfluidic mixing channel or by depositing and mixing two different reactants directly on the grid [1]. Although such techniques allow precise control over the initiation of chemical reactions in the time-resolved cryo-EM, they still suffer from uneven ice thickness and air-water interface issues [2]. We developed a microfluidic cryo-EM sample preparation technique where we freeze the whole microfluidic mixer channel that contains samples with varying reaction times. We fabricated a microfluidics cryofixation system on a cryo-stage with a microheater embedded in a thin parylene film to maintain the liquid sample at room temperature until the sample is ready for vitrification. After we successfully vitrified our sample, we cut out lamellae from the channel at different time points using cryo-FIB. Then the lamellae are transferred into cryoTEM to observe the structural changes that happen over time. This method allows the preparation of time-resolved cryo-EM samples that contain a wide range of reaction times and dynamic changes. We anticipate this system will allow the preparation of diverse types of biological samples for time-resolved cryo-EM studies including dynamics of biochemical reactions and changes in nanostructures of cellular organelles after certain external stimuli are applied.

References

[1] M. E. Mäeots et al. Nat. Commun., vol. 11, no. 1, pp. 1–14, 2020, doi: 10.1038/s41467-020- 17230-4.

[2] N. Alex J. et al., Nat. Methods, vol. 15, no. 10, pp. 793–795, 2018, doi: 10.1038/s41592-018-0139-3