Unrivalled performances

  • Higher yield of amenable cells for Cryo-ET experiments
  • Precise cell positioning within EM grids meshes
  • Multiple and standardized cells models
  • Undamaged surface thanks to maskless photopatterning

Higher yield of amenable cells for your Cryo-ET experiments

Cryo-ET is a powerful tool for imaging complex structure under close-to-life conditions. However, cell samples need some specific requirements to be imaged. One obvious advantage of EM grids micropatterning is the higher number of cells per grid that are amenable for further observation which makes the results of experiments more efficient. Thus, EM grids are not overcrowded since cells only adhere on the micropatterned area. It keeps cell density to a minimum, which increases the quality of the vitrification, while maximizing the number of cells in the mesh.

(a) Cryo-SEM of HeLa cells on a standard gold-mesh grid. Arrowheads indicate the cells optimally positioned for FIB-lamellae preparation. (b) HeLa cells on a gold-mesh holey grid with 20 μm diameter fibronectin micropatterns. (c-d) HeLa cells, expressing GFP-tagged β-tubulin (Cyan) and mCherry-tagged histone (Magenta), seeded on a (c) control and (d) patterned gold-mesh grids. Scale: 20 μm. M. Toro-Nahuelpan et al., Nature Methods, 2019

Precise cell positioning within EM grid mesh

Micropatterning allows flexible cell positioning, which is an asset for Cryo-ET by enabling to place the cells in the center of grid squares. It makes them perfectly accessible for the focused ion beam milling and imaging.

Methods in Microbiology. Léa Swistak et al, 2020

A) number of single cells positioned on the center of the gird mesh. 11 patterned grids and 8 non patterned grids. The Mann-Whitney nonparametric test was used to compare the distribution (P < 0.05). Application note, 2022.

Multiple shapes and standardized cells models

Primo allows you to choose the shape you want for your cells, and is very useful to target specific internal elements when correlative imaging approaches proves to be challenging. So, it appears as a perfect tool for conducting homogeneous and standardized Cryo-EM experiments.

Cytoskeleton study on live cells on EM grids patterned with PRIMO maskless micropatterning - M. Toro-Nahuelpan et al., BioRxiv, 2019
(a) On-grid live-cell confocal microscopy of actin organization in RPE1 LifeAct-GFP cells grown on micropatterns done with PRIMO (gold-mesh, SiO2 film R1/4). Yellow arrowheads: actin stress fibers. Blue arrowhead: actin rings composed of putative bundles. (h-i) SEM of a cell grown on a crossbow-shaped pattern done with PRIMO (yellow). P1 & P2 squares: positions of tomographic slices in (j) and (k). M. Toro-Nahuelpan et al., Nature Methods, 2019

By controlling the available adhesive surface and cell adhesion on EM grids, we can modulate the cell location and spreading to better study cell-cell junctions. The optimize spreading also allows here to skip the FIB milling step, easing the Cryo-ET process.

Lattice micropatterning improves throughput for cryo-ET of cell-cell contacts.
(C, D) Fixed endothelial cells (ECs) on EM grids blanket-coated (C) and micropatterned (D). Dashed line: Z-stack reslice and line profile of DNA (teal) and VEcad signals (green). (E) Lattice micropatterning increased the distance between nuclei and cell-cell junctions. N = 10 grids, 120 measurements for blanket-coated condition, N = 3 grids, 320 measurements for micropatterned condition. (F) ECs on micropatterned glass overlaid with fluorescence image to show micropattern (green). (G) EC cell-cell contacts can be below 500 nm, the thickness threshold for cryo-EM. Scale bars, 20 μm. Engel, Vasquez et al, BioRxiv, 2020

Undamaged surface thanks to maskless photopatterning

As a maskless and contactless photopatterning system, PRIMO can project your micropatterns in UV light on the surface of EM grids without compromising their integrity.

Maskless photopatterning of EM grids using PRIMO system. L. Engel et al., BioRxiv, 2019.
ECM protein micropatterns (done with PRIMO Alvéole)on EM grids. L. Engel et al., BioRxiv, 2019
Left: ECM protein micropatterns on holey carbon EM grids (200 mesh gold grid bars) done with PRIMO maskless photopatterning. Scale bar= 50 μm. Right: Epithelial PtK1 cells confined on rhodamine-fibronectin micropatterns on EM grids generated by PRIMO maskless photopatterning. Scale bar= 10μm. L. Engel et al., JMM, 2019.

Digging deeper into cellular mechanisms with micropatterning and cryo-ET

Watch this tech talk from Cell Bio 2020 to see how micropatterning and cryo-ET can accelerate research breakthroughs on what really goes on in the cellular machinery at the molecular scale!

Speakers: Drs. Pierre-Olivier Strale, Leeya Engel, Matthijn Vos, and Léa Swistak, PhD Student

Watch the replay
Screenshot Tech talk Alveole Cell Bio 2020

Advancing Cell Biology with Correlative Cryo-Microscopy

Pr. Elizabeth R. Wright (Morgridge Institute for Research University of Wisconsin-Madison).

Learning Objectives:
  • Discussion on results: infectious disease, neurosciences.
  • Review of needed equipment / techniques for successful whole-cell cryo-ET.
  • Tips for defining and optimizing important parameters for advanced whole cell cryo-microscopy workflow.
Watch the replay
Screenshot webinar no3 cryo-CLEM series

Webinar Micropatterning on EM grids

Webinar and Q&A with Dr Leeya Engel (Stanford university): “Micropatterning on EM grids: A strategy for improving cell cryo-ET workflow”, based on the outcomes of her paper (L. Engel et al., JMM, 2019) using PRIMO maskless photopatterning.

Webinar Nature sponsored by Alvéole- Leeya Engel - Micropatterning on EM grids: A strategy for improved cellular cryo-ET workflow. Using PRIMO technology by Alvéole.

Tutorial video: Micropatterning on TEM grids

Homogeneous micropatterns automatically positioned within the mesh of a TEM grid, without damaging its surface:

  • One repeating pattern
  • Different series of micropatterns
Tutorial video PRIMO micropatterning alignment on TEM grids

Do you have a question about your project of experiment with PRIMO?

Our research and application development team can help you set up or optimize your experimental protocols!

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