PRIMO micropatterning for cryo-ET
What difference does it makes to use PRIMO to prepare my cell samples for cryo-ET ?
PRIMO maskless micropatterning system allows to control cell adhesion. Therefore, it guarantees you will get several cells positioned precisely where you want them to be on the EM grid (usually in the middle of the square composing the mesh of the grid).
Futhermore, it gives you full control over the design of micropattern you create. As shown in some of our users research works, you can then control cell shapes, polarity, spreading, and that for:
- mechanobiology studies
- facilitating the FIB milling process
- ensuring reproducible and more efficient cryo-ET imaging
Is PRIMO micropatterning technique compatible with all EM grids ?
Up to now, we have tested different material of grids and we can confirm that PRIMO micropatterning system can be used with grid made of carbon film, SiO2 films, Silicon Nitride film with or without mesh. As a maskless micropatterning technique, it should be compatible with all classic EM grids. You can contact us to check whether your grids model is compatible.
Is it possible to test PRIMO with the specific references of TEM grids I use ?
We can propose several options for you to test PRIMO micropatterning system. You can contact us here to discuss your project of cryo-ET experiment with us and find the best solution for you to test PRIMO.
Can the system perform micropatterning through the film?
Yes it is possible to pattern with the UV through the grid film since it is transparent. If you want to pattern on the mesh, make sure the grid place on the motorized stage of the setup is placed upside down (side of the objective).
How can I be sure my micropatterns are aligned within the frame of the EM grid mesh?
Leonardo photopatterning software automatically detects the EM grid mesh as you can see in this tutorial video. It then positions your micropatterns within it and adjusts their size ratio so it perfectly fits. And, of course, Leonardo software displays a preview of the expected result, so you can always make manual changes before launching the micropatterning sequence.
How long is the process of preparing micropatterned EM grids?
You can produce from 5 to 15 micropatterned EM grids per day, from the anti-founling coating step to the protein incubation.
Handling EM grids is a delicate step, it seems that your process adds even more handling now. Do you have a solution for that?
Indeed, the micropatterning process adds more handling steps, but after a training period you will be able to perform the patterning process without damaging the grids. Furthermore, we have developed specific PDMS stencils which allow to place the grid on a coverslip (or else) and maintain it in position throughout the whole micropatterning process, reducing the grid handling.
What micropattern resolution can be achieved on an EM grid with your PRIMO system?
The resolution obtained with PRIMO maskless micropatterning system on EM grids is 1.2 μm, same as on glass.
Can I store my patterned grids before the step of seeding the cells and for how long?
Yes, before the adsorption of proteins, the prepared EM grids can be stored in PBS for one month.
What is the thickness of the PEG coating?
The PEG layer is approximately 5nm thick.
Which cells can be micropatterned on TEM grids?
Like for micropatterning on glass, classic mammalian adherent cell types can be patterned on TEM grids. Up to now, we can confirm our team and users have successfully micropatterned the following cell types: HeLa cells, fibroblasts, epithelial cells, MDCKII, PtK1.
What is the impact of micropatterning on the cell state, functions ?
By controlling the cell adhesion, micropatterning has an impact on the regulation of their functions and behavior. With more controlled and reproducible biochemical microenvironments in vitro, micropatterning allows to study living cells in more physiologically-relevant conditions (Thery, Journal of Cell Science, 2010).
Are there some papers using your technology for cryo-ET to which I could refer to ?
Yes, you can have a look at the following papers:
- “Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies” M. Toro-Nahuelpan et al., Nature Methods, 2019 (Julia Mahamid's lab, EMBL)
- “Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies“ L. Engel et al., JMM, 2019 (Beth Pruitt's lab, Stanford University, UC Santa Barbara)
- “Lattice micropatterning of electron microscopy grids for improved cellular cryo-electron tomography throughput” L. Engel, C. G. Vasquez et al., BioRxiv, 2020 (Alexander Dunn's lab, Stanford University)