65 the alignment of the stamp on specifi c microstructures constitutes a real challenge.
2. Deep UV photolithography and photomasks The advantages and drawbacks of deep UV photolithography through photomasks are directly correlated to its process. After coating the desired cell culture substrate with the anti-fouling polymer, a photomask, which is a robust and opaque stencil perforated with desired microscopic shapes, is placed over it. Deep UV light is then shined onto the top of the photomask to degrade the PEG layer on the areas matching the holes in the photomask. The solution-based adhesive protein is then added and adsorbs to the previously illuminated areas only. Here, the alignment issue and the possible damages caused to soft substrates by direct contact seem obvious. But when this technique is well performed it has the advantage of producing a large number of identical micropatterns. However, this effi ciency in micropattern production is only valuable if the micropattern designs have been previously optimised for a specifi c experiment and cell type. Otherwise, a new photomask needs to be ordered, delaying the progress of the project.
3. Maskless and contactless photolithography
In 2016, the team of Dr Vincent Studer from University of Bordeaux, CNRS, presented a new micropatterning method based on the use of a digital micro-mirror device (DMD), also used in video-projectors, which they called light induced molecular adsorption of proteins (LIMAP) [8]. This technology has, since then, been improved and commercialised by Alvéole as the PRIMO device.
PRIMO is an optical module in which the main components are a DMD and a UV source (λ = 375 nm). This module can be installed on all the most common inverted microscopes. A dedicated software programme (named “Leonardo”) permits the selection, alignment, adjustment and replication of a preview of one or several desired micropatterns, of 1.2 µm minimum resolution, on the cell culture substrate placed on the motorised stage of the microscope. Once a micropatterning sequence is set, the software initiates PRIMO to project UV light onto the substrate previously coated with an anti-fouling layer and in the presence of a specifi c photo-initiator (PLPP). The combined action of the UV and PLPP locally degrades the anti-fouling coating, in only a few seconds. The protein is then added and adsorbs on the illuminated areas only, creating the micropatterns.
As a maskless and contactless system, the PRIMO system solves the issues of fl exibility, alignment, substrate integrity and effi ciency, which improves prototyping and optimisation capabilities. Indeed, the micropattern image can be projected on any standard cell culture substrate (stiff or soft, fl at or micro structured, large or microscopic) and tested to get the best possible protein micropatterns for each specifi c experiment. Furthermore, as a photolithography technology PRIMO can also be used to fabricate 3D micro structured substrates (microfabrication and hydrogel polymerisation), combining structuration and functionalisation abilities in one unique tool.
Figure 4a. Left picture: Single fi broblasts adhering on fi bronectin micropatterns. Credit: Alvéole
Figure 4b. Right picture: Pancreatic islet: THUNDER Imager 3D Cell Culture EDoF reconstruction of an isolated human islet to experimentally examine the expression of IL-17, a proinfl ammatory cytokine, in individual human islet cells. The images have the following markers: Insulin (AF488, green), Glucagon (AF555, red) and IL-17 (AF647, magenta) and Hoechst (nuclei, blue). Courtesy of Matthias Von Herrath Lab at the La Jolla Institute of Immunology, La Jolla, CA., USA, for Leica Microsystems
disease modelling are conducted on soft substrates. Here there are no doubts that the contactless micropatterning system PRIMO is a real advantage for this field as it is compatible with substrates of low stiffness; as shown by Dr Parameswaran’s team from Northeastern University in its work on airway smooth muscle cell contractions using micropatterning on gels of different stiffnesses ranging from 0.3 kPa to 40 kPa [10]. Another unique benefit of this innovative technology, not yet discussed, is the ability to locally control the protein density and generate gradients; making it a powerful tool for studying chemotaxis mechanisms or the immune response [11]. The growing number of research projects using this maskless, contactless technology confirms that researchers need to continue improving their control capabilities over the cell microenvironment in vitro by employing such a technology [12].
Cryo-electron tomography: The challenge of the perfect sample
In his 2012 paper published in Proceedings of the National Academy of Sciences (PNAS), Alexander Rigort stated “cryo-electron tomography provides unprecedented insights into the macromolecular and supramolecular organisation of cells in a close- to-living state”. Indeed, cryo-electron tomography has proven to be a great advance in microscopy to better visualise and understand intra and inter-cellular mechanisms in their native state and at the molecular level, such as viral infection mechanisms for example.
Figure 5. Tomogram from research on tethering proteasomes to nuclear pore complexes. Courtesy of Dr Ben Engel, Max Planck Institute of Biochemistry, Martinsried, for Leica Microsystems
Figure 3. PRIMO micropatterning platform. Credit: Alvéole.
Micropatterning: A fancy tool or a real asset for cell biology?
The recent progresses in bioengineering and bio-functionalised substrates opens up new opportunities to improve our understanding and questioning of cellular processes, extra-cellular matrix roles and related diseases [9]. Depending on the complexity of the scientific question, cell biology studies are conducted on different scales, going from single cells to small cell assemblies, tissues and, for a few years, organoids.
Even if micropatterning can be used for generating spheroids and analysing cell population forces, it is most applicable for studies on the cell cytoskeleton, traction forces, cell migration or disease modelling which often require single cell isolation or precisely controlled cell-cell interactions. Many experiments on cell forces and
But cryo-ET is still a young microscopy technique and all the steps of its workflow are being intensely reviewed and improved by both academic laboratories and industrial companies. Almost every step of the workflow is critical but in the case of eukaryotic cells, 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 and being thin enough for transmission electron microscopy.
The ideal thickness for cryo-ET is estimated around 200 nm. As a comparison, depending on the cell types, the thickness of eukaryotic cells randomly spread on a fl at surface can range between 0,5 µm (podosomes and lamellipodia) to 10 µm (centre of the cell around the nucleus).
By controlling the cell adhesion, spreading and shape, micropatterning overcomes the issues faced at the very first step of this workflow. Indeed, without micropatterning, cells in culture medium are randomly seeded onto EM grids and tend to adhere on the metallic mesh of the grid rather than the carbon film, preventing the electron to go through it when performing the transmission electron microscopy. Furthermore, the random seeding of cells is also correlated with a limited spreading making it more difficult to find the target due to the thickness. On the contrary, micropatterning allows the control over their location and the increase of cell spreading, therefore facilitating the next steps for cryo-ET cell sample
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