Obtain better cell samples for your cryo-EM and cryogenic electron tomography experiments !

Cryo-EM is rapidly expanding for imaging intracellular molecules conformations. However, imaging cell samples with cryo-ET can be a challenge. Indeed, the cells often adhere on the metallic grid frame and therefore cannot be imaged in transmission electron microscopy.

Unlike other micropatterning techniques, PRIMO maskless and contactless micropatterning provides unique advantages for your TEM and cryo-ET imaging experiments:

  • Cell positioning: precise automatic alignment within the mesh of EM grids!
  • Standardized cell models: reproducible micropatterning for mechanobiology studies
  • Undamaged surface: contactless micropatterning preserving the EM grid surface integrity

Cell positioning within EM grid mesh

Leonardo software detects the mesh of EM grids and automatically aligns your patterns within it, for PRIMO to perform aligned micropatterning. The PRIMO system thus ensures cells will be well positioned within the mesh of your EM grids !

(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., BioRxiv, 2019

And thus make sure you get cells optimally positioned for FIB milling and cryo-ET !

Toro-Nahuelpan et al., BioRxiv, 2019
(e) FIB shallow angle view on cell framed in (b). (f) Final lamella. (g) Tomographic slice, 6.8 nm thickness, of the nuclear periphery of the cell in (e). NPC: nuclear pore complex; MT: microtubule. M. Toro-Nahuelpan et al., BioRxiv, 2019

Micropatterning for standardized cell models

Micropatterning is known as an effective way to control cell adhesion in vitro. This technique is therefore largely used for standardizing cell shape as well as cell internal organization (directly linked to external adhesive cues).

With its unique capacity to automatically align micropatterns within the mesh of EM grids, PRIMO micropatterning appears as the perfect tool for conducting homogeneous and standardized cryo-EM experiments.

To go further, using PRIMO micropatterning for cryo-ET experiments on cells could help precisely target specific internal elements when correlative imaging approaches prove to be too challenging, as emphasised by M. Toro-Nahuelpan et al.*

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., BioRxiv, 2019
Cryo ET cytoskeleton study on micropatterned cell. M. Toro-Nahuelpan et al., BioRxiv, 2019
(j-k) Cryo-ET imaging: Tomographic slices of the positions 1 and 2 indicated in (h). Actin bundles likely equivalent to actin transverse arcs (parallel to but distant from the cell edge) and internal stress fibers -indicated in (a)- are found in locations expected according to the actin map in a crossbow-shaped RPE1 cell. M. Toro-Nahuelpan et al., BioRxiv, 2019

EM grid undamaged surface

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.
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., BioRxiv, 2019.

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

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