Obtain better cell samples for your cryo-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, before even considering going to the microscope, the cell sample must meet some basic requirements: matching specific locations on the EM grid, containing the target component, being thin enough for transmission electron microscopy.

By controlling the cell adhesion, spreading and shape, PRIMO maskless and contactless micropatterning overcomes the issues faced at the very first step of the workflow of cell sample preparation for cryo-ET.

It provides unique advantages for your whole-cell TEM and cryo-ET experiments:

  • Precise cell positioning: automatic alignment within the mesh of EM grids!
  • Optimized cell spreading: reduced thickness for more efficient CLEM and cryo-ET workflows
  • 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., Nature Methods, 2019

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

Toro-Nahuelpan et al., Nature Methods, 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., Nature Methods, 2019
Micropatterned neurons on EM grid - Elizabeth Wright
Maskless photo micro-patterning and cryo-EM of primary neurons derived from Drosophila melanogaster larvae. (A) Live-cell fluorescence microscopy of Drosophila neurons expressing membrane-targeted GFP on photopatterned grid squares with fluorescent concanavalin A. Green: Drosophila neurons. Light blue: Micropatterns. (B, C) Magnified live-cell imaging view of two regions from (A) showing ordered arrangement of the neurons along the photopatterned regions. (D) Cryo-EM grid low magnification map of the same grid in (A) after plunge-freezing. Green circle indicates the same grid square imaged in (E). (E) Higher magnification images collected of the neurites. (F) Cryo-EM image acquired from a region of the green circle in (D). Various structures can be seen in the image, such as the cell membrane, microtubules, vesicles, and actin. Courtesy of Joseph Y. Kim, Bryan S. Sibert, Jie E. Yang and Elizabeth R. Wright. Department of Biochemistry & Cryo-Electron Microscopy Research Center, University of Wisconsin, Madison. Morgridge Institute for Research, Madison.

Optimized cell spreading on EM grids thanks to micropatterning

By controlling the available adhesive surface and cell adhesion on EM grids, PRIMO micropatterning can modulate the cell spreading. And the cell spreading in turn can be a key asset for improved throughput for whole-cell cryo-electron tomography. Indeed, the spreading facilitates the identification (CLEM), preparation (FIB milling, sometimes not even needed if the sample is thin enough) and imaging (cryo-ET) of the intra-cellular targeted structures.

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

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., Nature Methods, 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., Nature Methods, 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., JMM, 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.

Ressources: Using the PRIMO micropatterning system for cryo-ET

Find below several support tools to help you further investigate the use of the PRIMO micropatterning system to facilitate cryo-ET experiments.

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

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