Puffalot is a unique device for the preparation of cryo-electron microscopy (cryo-EM) samples that uses a puff of compressed gas to remove excess material from the sample grid, leaving only a thin layer suitable for cryo-EM imaging, in a consistent, reproducible manner.
Traditional plunge-freezers use filter paper to remove excess sample, leading to variable quality in the resulting sample grids. Dr Meindert Lamers’ research group at MRC LMB developed Puffalot to solve this problem and create a more reproducible method. Puffalot uses a puff of gas to remove excess liquid from the sample grid. Using a gas stream provides finer control over the quality of the sample and gives more reliable results. Puffalot features a video camera that monitors and records the sample grid during preparation, providing immediate feedback about its quality. With traditional methods the filter paper blocks the view of the grid which can only be assessed once mounted into a cryo-electron microscope, hours or even days after preparation.
This technology is the subject of patent application: PCT/EP2017/069240.
Puffalot’s key features
- a unique plungefreezer device for cryo-EM
- offers greater quality and reproducibility in sample preparation
- monitors sample preparation using a high-resolution camera
- existing prototype available
Current methodologies for preparing cryo-EM samples rely on filter paper to blot away excess liquid from the cryo-EM grid, which can result in variation in the thickness of the protein sample and in compromised image quality. Typically, only one or two ‘good’ grids are achieved for every 10 attempted. Samples can only be evaluated in the electron microscope, an instrument that is not suitable for high throughput analysis. Consequently, sample grid preparation is one of the most significant bottlenecks in cryo-EM structure determination. Puffalot provides a more reproducible method for preparing sample grids and methods to determine the quality of the sample grid before testing, without a cryo-electron microscope.
Advantages over existing methods
- The pressure and timing of the puff of gas can be accurately defined giving more reproducible results
- The directionality of the gas stream allows for fine control of the thickness of the sample layer over the width of the grid
- Without filter paper, a high-resolution camera can be used to monitor and record the grid, and the sample, during preparation. This allows the sample to be monitored from preparation through to the final grid observed in the cryo-electron microscope, allowing adjustments to be made
- Adverse interactions between the macromolecule and the filter paper, and with the air-water interface, are avoided
With Puffalot, grid preparation is not a ‘hit-and-miss’ procedure but an accurately defined technique that takes the uncertainty out of sample preparation allowing the efficient use of scarce cryo-EM resources. Puffalot is much faster than existing products and prototypes.