Patent Application
20220301810
The invention relates to the field of preparing biological specimen for microscopic analysis, particularly for the electron microscopic (EM) analysis of cells on respective EM supports. The invention particularly relates to electron microscopy (EM) supports for in situ cryo-electron tomography, particularly to contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins for micromachining by cryo-focused ion beam milling. The new EM supports allow for analysis of intracellular organization, permitting direct correlation of cell biology and biomechanics by 3D-structural characterization of the underlying molecular machinery in cellulo.
Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for in situ cryo-electron tomography. Here, we describe a contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex patterns generate predictable intracellular organization, allowing direct correlation of cell biology and biomechanics by 3D-structural characterization of the underlying molecular machinery in cellulo.
In parallel to the ongoing resolution revolution in cryo-electron microscopy for macromolecular structure determinationRef.1, cryo-electron tomography (ET) has developed further to reveal the molecular sociology in situ sensu strictoRef.2-4. Yet, cryo-ET of adherent cells, particularly mammalian cells, can only be directly performed on their thin peripheries (<300 nm). To reveal collective behaviors of macromolecules at the cell interior, thinning by advanced cryo-focused ion beam (FIB) has proved an optimal, artifact-free preparation methodRefs.2,5,6. Specimen preparation for cellular cryo-ET, whether performed directly on thin cellular peripheries or following FIB micromachining, routinely involves seeding of adherent cells directly on EM grids. Standard EM grids are 3 mm diameter metal meshes overlaid with a delicate perforated thin film. Cells are typically allowed to spread, subjected to genetic or molecular perturbation to represent different physiological settings to be examined in molecular detail, that are then arrested by vitrificationRef. 7. For cells to be thinned by cryo-FIB, they must be positioned roughly at the center of an individual grid square (
The present invention combines cellular cryo-ET with another technology in the fields of cell biology and biophysics, that of spatially controlled cellular environments. By adaptation of photo-micropatterning routinely applied to centimeter-scale glass slides for light microscopy-based assaysRefs. 8,9, functionalized EM supports were developed for directing cell positioning at high spatial accuracy, which ultimately renders molecular-resolution imaging of frozen-hydrated specimens more easily attainable.
Therefore, it was an objective of the present invention to provide means and methods that overcome the above problems associated with the analysis of biological specimen by way of electron microscopy. Other problems that have been solved and advantages associated herewith will be understood upon reading the detailed description and the experimental part of this disclosure.
Generally, and by way of brief description, the main aspects of the present invention can be described as follows:
In a first aspect, the invention pertains to a functionalized electron microscopy (EM) support comprising at least one or more area(s) functionalized with substrate allowing for the adhesion, generally speaking for the immobilization, of biological specimen, particularly live cells, wherein the area(s) is/are at least partially, preferably completely, surrounded by a passivation layer substance, wherein said substance repels cells, particularly mammalian live cells, or even does not allow for the adhesion of said live cells.
In a second aspect, the invention pertains to a method of producing the electron microscopy (EM) support referred to in the first aspect.
In a third aspect, the invention pertains to uses of the EM support forming the first aspect and that have been prepared by the methods according to the second aspect. Generally spoken, these uses relate to methods of analysing the biological specimen, e.g. adherent mammalian cells using a variety of microscopic methods, particularly EM-based methods.
In a fourth aspect, the invention pertains to a method for producing a circuit of cells.
In a fifth aspect, the invention pertains to the use of the circuit of cells in medicine.
In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
In a first aspect, the invention pertains to a functionalized electron microscopy (EM) support comprising at least one or more area(s) functionalized with at least one or more substrate(s) allowing for the adhesion of biological specimen, particularly live cells, or generally for the immobilization of macromolecules, such as biological macromolecules, for example antibodies, hormones, toxins, cytokines, etc., wherein the area(s) is/are at least partially, preferably completely, surrounded by passivation layer substance, wherein said substance repels live cells and/or essentially does not allow for the adhesion of live cells. Alternatively, the passivation layer substance is a substance that prevents adhesion of live cells by at least 50% or more compared with areas that are functionalized with at least one or more substrate(s) allowing for the adhesion of biological specimen, particularly live cells.
In a second aspect, the invention pertains to a method of producing the electron microscopy (EM) support according to the first aspect.
In a third aspect, the invention pertains to uses of the EM support forming the first aspect and that have been prepared by the methods according to the second aspect.
In a fourth aspect, the invention pertains to a method for producing a circuit of cells.
In a fifth aspect, the invention pertains to the use of the circuit of cells in medicine.
The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and/or claimed herein.
As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.
It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.
Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.
In view of the above, it will be appreciated that the present invention also relates to the following itemised embodiments:
In a first item, the present invention relates to a functionalized electron microscopy (EM) support comprising at least one or more area(s) functionalized with at least one substrate allowing for the adhesion of biological specimen, particularly live (mammalian) cells, particularly live cells, or generally for the immobilization of macromolecules, such as biological macromolecules, for example antibodies, hormones, toxins, cytokines, etc., wherein the area(s) is/are at least partially, preferably completely, surrounded by at least one passivation layer substance, wherein said substance repels live cells and/or essentially does not allow for the adhesion of live cells. Alternatively, the passivation layer substance that prevents adhesion of live cells by at least 50% or more, for example by at least 60%, by at least 70%, by at least 80%, by at least 90% compared with areas that are functionalized with at least one or more substrate(s) allowing for the adhesion of biological specimen, particularly live cells. For the analysis of immobilized macromolecules the passivation layer substance has also the effect that the macromolecules that are analysed tend to be immobilized in the area(s) that have been treated to the passivation layer and that have been functionalized to immobilize the macromolecules referred to above. The ablation of the passivation layer substance is preferably made with a pulse-laser suitable for photo-micropatterning the support, e.g. an EM grid.
The invention hence relates to electron microscopy (EM) supports for in situ cryo-electron tomography, particularly to contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins for micromachining by cryo-focused ion beam milling. The new EM supports allow for analysis of intracellular organization, permitting direct correlation of cell biology and biomechanics by 3D-structural characterization of the underlying molecular machinery in cellulo.
In further items, the biological specimen may be any biological macromolecule of interest that may be positioned in the micro-patterned area(s). Examples are proteins including antibodies, which may be of assistance in, for example, Single Particle Analysis (SPA). Micro-patterned areas can be modified chemically as long as this chemical component does not also bind to the passivation layer substance by at least 50% or more, for example by at least 60%, by at least 70%, by at least 80%, by at least 90% compared with areas that are functionalized with at least one or more substrate(s) allowing for the adhesion of biological specimen.
In a further embodiment, the functionalized EM support is based on an electron microscopy grid, for example a grid comprising or consisting of gold, copper, molybdenum, or titanium, optionally comprising a biocompatible layer, preferably a SiO2-, graphene-, carbon-, or gold-film, particularly a SiO2-film or a graphene-film. As used herein, a grid is a metal mesh (i.e., it looks like crossed bars) with a thin film on top in the whole grid. The film is usually patterned in the middle of a grid square.
In accordance with other embodiments of the invention, it is also possible to create a pattern on top of the grid bars. This permits using interacting partners (Streptavidin in the patterns and Biotin in the fiducials, or Antibody anti-GFP in patterns and GFP on fiducials) which can be directed to the regions of the film that are on top/above the grid bars, e.g., fiducials may be positioned on top of such bars, which is of major importance for correlation purposes between light and electron microscopy.
Therefore, in a further embodiment, the functionalized EM support further comprises fiducials, particularly, fluorescent beads specifically positioned on the EM support, particularly on top of grid bars, which may be used, for example, for the above-indicated purposes. A particularly preferred embodiment relates to micropatterning of fiducials, particularly, fluorescent beads to bars.
In a further embodiment, the functionalized EM support comprises a passivation layer substance that comprises or is a repelling agent, e.g. a biochemical or biophysical repelling agent. As used herein, repelling means that the biological specimen essentially do not bind to/adhere to the substance forming the passivation layer, or that the binding/adhesion is reduced by at least 50%, preferably more, as pointed out above. The passivation layer may be polyethylene glycol (PEG). Particularly, two different passivation examples are described in the following. In one approach, PLL-PEG may be used, where the PLL section binds to the surface and exposes the PEG chains forming initially coated layer that is subsequently ablated. In another approach a two-step passivation may be performed by first treating EM grids with PLL alone and subsequently with PEG-sva that binds covalently to PLL. It is also possible to use a bio-passivation agent such as protein or DNA that may be ablated with the laser, leaving few micro-patterned areas, where the biological agent is used as bio-passivation. Also, detergents, such as Tween, could be used to passivate surfaces, but they are not biocompatible, i.e. cells will not grow there. Such passivation can be useful in proteomic analysis, e.g. transmembrane proteins (reference is made to the above-mentioned Single Particle Analysis of biological macromolecules). In particular embodiments of the present invention, the tools and methods described herein pertain to the analysis of cells, e.g. mammalian cells, but cells from organisms other than mammals, or even microorganisms are also suitable for analysis using the herein described EM supports.
In further items of the present invention, the passivation layer substance forms a pattern comprising one or more microscopically distinguishable area(s) so that defined areas comprising the cells or macromolecules of interest can be distinguished. Microscopically distinguishable means that the respective areas may not always be distinguishable to the naked eye, but also that the areas may not be absolutely identical in shape and size. However, these areas should appear essentially homogenous and regular when viewed through a microscope.
In further items, the repelling agent comprises a polyether, polyethylene glycol, and/or PLL-g-PEG as explained in more details in the examples section.
In further items, the substrate for the adhesion of live cells comprises proteins, glycoproteins, and/or polysaccharides, particularly proteins are suitable as substrates for live cells. In embodiments of the invention, the substrate for the adhesion of live cells comprises at least one extracellular matrix component selected from the group comprising laminin, fibronectin, vitronectin, integrin, collagen, fibrillin, elastine, glycosaminoglycane and the like.
In further items, when biological macromolecules such as antibodies, etc. are analysed, the functionalization substrate may be adequately selected to ensure the specific binding to areas in the grid that are sufficiently distant from the passivation layer so that the molecules can be relatively easily distinguished and characterized.
In further items, the present invention relates to the functionalized electron microscopy (EM) support comprising at least one live cell or at least one fixed cell per one area. The cells may still be capable of cell division before fixation (vitrification) which may increase the number of cells per given area. In some embodiments, the cells may be selected from the group comprising stem cells, induced stem cells, pluripotent stem cells, primary cells, transformed cells, neuronal cells, blood cells, immune cells, cancer cells, genetically engineered cells, infected cells, and the like. In some embodiments, the cells are mammalian cells, wherein the term “mammalian” comprises any mammal, particularly humans. However, it is also possible to use cells that are not of mammalian origin, e.g., cells derived from insects, fish, birds, etc.
In still further embodiments, the present invention relates to a method of preparing the functionalized electron microscopy (EM) support according to any of the foregoing embodiments for specimen analysis, said method comprising:
In further items, the present invention relates to the method according to the preceding embodiment, wherein the photo-micropatterning step is a contactless and/or mask-free photo-micropatterning step.
In further items, the present invention relates to the method according to any one of the preceding items, the photo-micropatterning step removes or ablates the passivation layer substance of step b). Step b) may be preceded by a cleaning step, e.g. a plasma cleaning step in order to render the grids oxidized and hydrophilic before coating the same the passivation layer substance.
In further items, the present invention relates to the method according to preceding embodiments, wherein the photo-micropatterning step is performed with a UV-laser, particularly a 300 nm to 370 nm pulse laser, 310 nm to 370 nm pulse laser, 320 nm to 365 nm pulse laser, 330 nm to 360 nm pulse laser, particularly a 355 nm pulse laser. Alternatively, the photo-micropatterning step is performed by UV-illumination with a digital micro-mirror device (DMD). When using a laser at less than 300 nm or above 375, the ablation of the passivation layer does not function properly or, at less than 300 nm, cause stronger damage to the EM support.
In further items, the present invention relates to the method according to preceding embodiments, further comprising step d) comprising functionalizing the previously ablated (i.e. passivation layer-free) areas of the of the EM support with at least one substrate allowing for the adhesion of live cells in those areas where the photo-micropatterning step removed the passivation layer substance applied in step b). When the specimen subject to analysis is a biological macromolecule, the substrate may also be a chemical substrate allowing for the specific binding of the macromolecule to the functionalized are, wherein the macromolecule essentially does not bind or adhere to the passivation layer substance(s).
In further items, the present invention relates to the method according to preceding embodiments, wherein the passivation layer substance comprises or is a repelling agent. As indicated above, the passivation layer substance preferably forms a pattern of one or more microscopically distinguishable fields, particularly a grid-shaped pattern. The repelling agent may comprise a polyether, particularly polyethylene glycol.
In further items, the present invention relates to the method according to preceding embodiments, wherein the substrate for the adhesion of live cells comprises proteins, glycoproteins, polysaccharides, amongst other.
In further items, the present invention relates to the method according to preceding embodiments, further comprising at least one step d), wherein at least one live cell is seeded in at least one area functionalized with a substrate allowing for the adhesion of live cells. Alternatively, it is possible to functionalize the respective fields with a substrate that is specifically adapted to bind biological macromolecules.
In further items, the present invention relates to the method according to preceding items, wherein said method further comprises at least one step e), wherein at least one or more live cell(s) is/are fixed to the EM support, precisely to the substrate-functionalized areas.
In further item, the present invention relates to the method according to any of the preceding items, wherein said method comprises automated cryo-FIB milling.
In further embodiments, the present invention relates to the use of an EM-support as defined in any one of preceding embodiments or of an EM-support prepared in a method according to any one the above embodiments in the (e.g. structural and biophysical, biomechanical) analysis of adherent cells (for example, cell shape, cytoskeletal architecture, stereotypical internal organization of cellular organelles, etc.), particularly comprising at least one method selected from the group comprising microscopy, confocal microscopy, vitrification, cryo-FIB milling, transmission electron microscopy, cryo-light microscopy, cryo-electron tomography, cryo-focused ion beam (FIB) analysis, cryo-correlative light-electron microscopy (Cryo-CLEM), and/or cellular micromachining by cryo-FIB milling.
Yet another aspect of this invention, which can be combined with any other aspect and/or embodiment of this invention, pertains to a method for producing a circuit of cells, comprising the steps of:
According to this invention, the term “a circuit of cells” shall refer to any device capable of holding at least two cell types, such as a chip, wherein said device comprises at least two cells growing in form of a micropatterned-based circuit, for example in form of a micropatterned-based circuit on a grid.
Another specific embodiment of this invention relates to the method for producing a circuit of cells, wherein the cells are selected from neurons, hepatocytes, myocytes, cardiomyocytes, stem cells, stem cell progenitor cells, trophoblasts, astrocytes, glial cells, enterocytes, hepatic cells, kidney cells, endothelial cells, epithelial cells, such as biliary epithelial cells, syncytiotrophoblasts, cytotrophoblasts, mesenchymal cells, inner cochlea cells, outer cochlea cells, trophoblasts, preferably wherein the cells are human cells, such as human neurons.
A further preferred embodiment of this invention relates to the method for producing a circuit of cells, wherein said cells belong to the same cell type or to at least two different cell types. Preferably, the method for producing a circuit of cells is used to generate a circuit of cells belonging to the same cell type or belonging to different cell types. The latter can, for example, be a circuit comprising different brain cells, such as neurons, astrocytes and glial cells.
A further specific embodiment of this invention relates to a method for producing a circuit of cells, further comprising the step of:
Yet another aspect of this invention, which can be combined with any other aspect and/or embodiment of this invention, pertains to a circuit of cells produced by the method for producing a circuit of cells of the specific aspect and embodiments above.
A further aspect of this invention relates to a circuit of cells produced by the method for producing a circuit of cells of this invention for use in medicine.
Yet another aspect of this invention, which can be combined with any other aspect and/or embodiment of this invention, pertains to a circuit of cells produced by the method for producing a circuit of cells for use in the treatment and/or prevention of a brain disease, spinal-cord injury, a heart disease, liver failure, kidney failure, deafness, a degenerative disease, such as a neurodegenerative disease, and/or a skin disease, or in the manufacture of a medicament against a brain disease, spinal-cord injury, a heart disease, liver failure, kidney failure, deafness, a degenerative disease, such as a neurodegenerative disease, and/or a skin disease.
Another aspect of this invention relates to a circuit of cells produced by the method for producing a circuit of cells of this invention to repair at least one damaged circuit in or on the human body, for example to repair a damaged neuronal circuit. According to this aspect of the invention, said circuit of cells produced by the method for producing a circuit of cells can be used as an implant or as a pacemaker, preferably as an organ implant, such as a brain implant, a cochlea implant, or a liver implant.
Yet another aspect of this invention pertains to a method of treatment and/or prevention of a brain disease, spinal-cord injury, a heart disease, liver failure, kidney failure, deafness, a degenerative disease, such as a neurodegenerative disease, and/or a skin disease in a subject, the method comprising the step of administering to the subject a circuit of cells produced by the method for producing a circuit of cells of this invention.
A further preferred embodiment of this invention relates to the method of treatment and/or prevention, wherein said subject is a mammal, such as a human, a mouse, rat, guinea pig, rabbit, cat, dog, monkey, preferably a human, for example a human patient, more preferably a human patient suffering from a brain disease, spinal-cord injury, a heart disease, liver failure, kidney failure, deafness, a degenerative disease, such as a neurodegenerative disease, and/or a skin disease.
Yet another aspect of this invention pertains to the use of a functionalized electron microscopy support defined in any one of preceding embodiments or of a functionalized electron microscopy support prepared in a method according to any one the preceding embodiments for generation of a circuit of cells on the electron microscopy support.
A further aspect of this invention pertains to the use of a functionalized electron microscopy support defined in any one of preceding embodiments or of a functionalized electron microscopy support prepared in a method according to any one the preceding embodiments as a neuronal microprocessor for computing, information processing and storage.
Additional items of the present invention:
Other embodiments will become apparent to the person skilled in the art in light of examples infra.
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.
In order to perform the present invention contactless and mask-free photo-micropatterning were adapted (
HeLa cells are a prominent model system in cell biology, and must be thinned to reveal structures positioned deep in their interior by cryo-ET. We seeded HeLa cells on fibronectin-functionalized micropatterned (30 μm disk-shape) grids. A reproducible seeding of single or double cells at the center of individual grid squares was achieved (
Next, complex patterns to control cell shape were generated. Micropatterning on glass surfaces has been previously shown to induce well-defined cytoskeletal architectures and, as a result, a stereotypical internal organization of cellular organellesRefs. 8,9.
Here, the actin network in Retinal pigment epithelium cells (RPE) as a case of study and as a direct readout of the cellular response to adhesion on the complex patterns will be described. Tailored micropatterns induced reproducible cell morphology on grids (
To explore the organization of the cytoskeleton further away from the cell peripheries and to characterize spatially-predictable structures according to live-cell actin maps (
In conclusion, photo-micropatterning of EM grids contributes to the advancement of refined, routine and user-friendly specimen preparations for in-cell structural biology. It further aids in solving technical challenges that have, thus far, hindered high-throughput FIB thinning preparations. This method will be instrumental for potential automation of the cryo-FIB milling process, deeply impacting the streamlining state-of-the-art cellular cryo-EM pipelines. This approach offers a unique opportunity to generate in-cell integrated insight into the structure and dynamics of macromolecules at nanometer-scale, broadening the scope of questions that can be addressed by state-of-the-art structural biology methods.
Methods
Cell Lines and Culture
Wild type HeLa Kyoto cells, and a double tagged line expressing both green fluorescent protein (GFP)-tagged β-tubulin from a bacterial artificial chromosome (BAC) and mCherry tagged histone from a plasmid construct (H2B-mCherry). HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM; ThermoFischer Scientific, Schwerte, Germany), while RPE-1 (Retinal Pigment Epithelial human cells) expressing LifeAct-GFP19 were cultured in DMEM F-12. Cells were incubated at 37° C. with 5% CO2, and supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 mg/mL penicillin, 100 mg/mL streptomycin. A 0.5 mg/mL geneticin (G418) for the BAC-tagged lines and Puromycin (1 μg/ml) for cells carrying the plasmids were used. FluoroBrite DMEM (ThermoFischer Scientific, Schwerte, Germany) was used for live cell fluorescence imaging.
Electron Microscopy Grids
Gold (Au) or Titanium (Ti) 200-mesh grids with a holey 12 nm thick SiO2 film, either R2/1, R1/4 or R1/20 (Quantifoil Micro Tools, Jena, Germany) were employed in this study. Titanium-mesh grids, and SiO2 films replacing the commonly used amorphous carbon Quantifoil, provided stiffer and more robust supports for the multiple grid processing and cell culture steps described in the method. Both, titanium (
Increased amount of film over holes promoted better cell adhesion. R2/1 and R1/4 films were advantageous for direct tomography of peripheral cellular areas, while R1/20 and R1/4 films were more suitable for cellular thinning by cryo-FIB milling as the majority of the film is removed during thinning.
Grid Passivation
One-step: Grids were oxidized and rendered hydrophilic using a low-pressure Diener Femto Plasma cleaner. Grids were place onto a glass slide and both sides were plasma cleaned at 100 W power with a flow rate 10 cm3/min of oxygen gas for 30-40 s. Next, grids were incubated on droplets of poly(l-lysine) grafted with poly(ethylene glycol) (PLL(20)-g[3.5]-PEG(5), SuSoS AG, Dübendorf, Switzerland) at a concentration of 0.5 mg/ml in 10 mM Hepes pH 7.4, for 1 h at room temperature or overnight at 4° C., on a parafilm in a humid chamber (parafilm sealed dish with soaked filter paper). Following passivation, the grids were blotted with filter paper from the side and allowed to dry. No washing of the PLL-Peg was performed.
Two-step: As an alternative, a two-step treatment of the grids was also tested. First, grids were incubated on droplets of 0.01% PLL (Sigma Aldrich, St. Louis, Mo.) on a parafilm in a humid chamber overnight at room temperature. Next, the grids were blotted, but not allowed to dry and incubated for 1-2 h with 50 mg/ml PEG-sva (Laysan Bio, Arab, US).
Both passivation protocols were successful for grid passivation followed by photo-micropatterning, as judged from fluorescence light microscopy imaging of GFP-absorption that was restricted to the PEG-free patterns. However, a one-step PLL-g-PEG passivation was preferred for time optimization purposes. This treatment is especially convenient in the absence of plasma cleaner and can be used following the more commonly employed glow discharge procedures.
Micropatterns were designed in Inkscape (http://www.inkscape.org/) as 8-bit binary files and exported as png files, which can be loaded into the Leonardo software (Alveolé Lab, Paris, France).
Micropatterning and Functionalization of Grids
Nanoablation by a 355 nm Pulse Laser
An inverted confocal Olympus FluoView 1200 (Olympus, Hamburg, Germany) microscope was used, equipped with a UV pulsed laser source of 355 nm (PNV-001525-140, Teem Photonics, Meylan, France), a UPLSAPO 63× (NA 1.35) oil objective, and a standard PMT or GaAsP PMT detectors. The 355 nm laser had an average power of 50 mW, 300 ps pulse width, 1 kHz repetition rate, and a maximum energy per pulse of 20 μJ. Transmission and reflection were observed with a 488 nm laser. The patterns (ROIs) were of circular shape (20, 30 or 40 μm diameter) and made using the Olympus FV 10-ASW software v04.02.03.02. Photo-micropatterning was performed using 10-11% laser power, 40 82 s per pixel and 10 iterations. Individual grid squares were targeted at a time, the film focused and the laser applied. Micropatterning of a 4×4 grid square area (200-mesh grid: ˜260,000 μm2) took ˜8 min. Potentially, a lower magnification objective can be used in order to pattern more grid squares at the same time in order to optimize patterning, provided that the film is flat and at even height to maintain all areas in the focus plane. Titanium grids had a consistent film flatness aiding quick focusing on each grid square, facilitating the micropatterning using this technique.
PRIMO™ (DMD-Based Illumination+Photo-Activator):
An inverted Nikon microscope Ti-E equipped with a CFI Super Plan FLuor 20× ELWD (NA 0.45) lens with high UV-transmission, a Perfect Focus System 3, an ORCA-Flash 4.0 LT CMOS camera (Hamamatsu, Japan), a motorized stage (Märzhäuser, Wetzlar, Germany), and the Primo™ micropatterning module (Alveolé Lab, Paris, France) was used. Grid micropatterning was performed using digital mirror device (DMD) to generate a spatially controlled laser illumination of the sample (Primo™, Alveolé Lab, Paris, France), which provides a resolution limit of ˜1.2 μm. After passivation with PLL-g-PEG, grids were blot-dried from the back with a filter paper and quickly placed with the SiO2 film facing up (away from the objective) on a 1-3 μl of PLPP (4-benzoylbenzyl-trimethylammonium chloride, 14.5 mg/ml) drop in a sealed glass bottom ibidi μ-Dish 35 mm low (ThermoFischer Scientific, Schwerte, Germany). High humidity was kept using water-soaked filter paper inside the dish to avoid PLPP evaporation. The dish, with 1-4 grids at a time, was placed on the microscope stage and photo-patterning was controlled with the μmanager software v1.4.22 by the Leonardo plugin software v4.12 (Alvéole Lab, Paris, France) using the stitching mode and a 375 nm (4.5 mW) laser, applying a dose of 800-1000 mJ/mm2 equivalent to ˜30 s per DMD exposition. Micropatterning of an 8×7 grid square area (200-mesh grid: ˜900,000 μm2) took 3-7 min depending on the total dose and grid positioning with respect to the DMD mirror illumination. Grids were promptly retrieved from the PLPP solution, washed in a 300 μl drop of water, and two consecutive washes in 300 μl drops of PBS. Grids were stored wet in PBS at 4° C. in a humid chamber, remaining functional for at least 30 days.
For functionalization following PEG ablation in micropatterns using both methods, grids were incubated at room temperature in a 20 μl drop of either 50 μg/ml fibronectin (ThermoFischer Scientific, Schwerte, Germany), a 50 μg/ml of a GFP-tagged protein, or 50 μg/ml of fibrinogen-488 (ThermoFischer Scientific, Schwerte, Germany) on a parafilm and, subsequently, washed 3 times in 300 μl drops of PBS. Grids incubated with fibronectin remain functional for at least to 10 days in PBS at 4° C. in a humid chamber. The maximum active life time of the micropatterned grids as well as protein functionalization remain unknown. This will also depend on the protein stability itself. All patterning steps and grid treatments were performed under sterile conditions using a Bunsen burner. Grids were handled with a tweezer n° 55 (Dumont, Montignez, Switzerland).
Comparison Between the Two Patterning Approaches:
Due to the pulsing nature of the laser in the first approach, it has to scan the region of interest to be patterned to ablate the anti-fouling agent. The action of the laser leaves an impression on the film that is visible by light microscopy (
While the Primo device takes 30 s per DMD run (for a dose of 1000 mJ/mm2 and covering a 3×2 grid squares on a 200-mesh grid), the 355 nm-pulse laser patterning takes ˜10-15 s per grid square considering a disk-shaped pattern of 20-30 μm diameter. A user familiar with the 355 nm laser technique can pattern a 4×4 grid square area in ˜8 min, while the Primo technology covers a similar area in ˜1 min. While the Primo device is faster to create micropatterned areas, the 355 nm-pulse scanning laser can yield a much higher spatial lateral resolution limited by the light diffraction (PSF) and equivalent to ˜250 nm, in comparison to the Primo performance that is limited to ˜1.5 μm.
At least 50 grids have been seeded with either HeLa or RPE cells obtaining reproducible results with cells settling and adhering to the micropatterned areas.
Cell Seeding
Non-patterned grids were plasma cleaned or glow discharged. Cells were detached from cell culture flasks using 0.05% trypsin-EDTA and seeded on pre-treated Quantifoil grids in glass bottom ibidi μ-Dish 35mm high (ThermoFischer Scientific, Schwerte, Germany).
Cells were seeded on fibronectin micropatterned surfaces right after being passed through a cell 40 μm pore-size cell strainer (Corning, Amsterdam, Netherlands) at a density of 2×104 cells/cm2 for HeLa and 8×103 cells/cm2 for RPE cell lines. After seeding, grids were incubated for 1.5-2 h for HeLa cells or 20-35 min RPE cells. Next, cells were transferred to a new cell-free dish and incubated at 37° C. with 5% CO2 to allow adhesion to the grids. Transfer to a new dish was beneficial to remove cells that were non-specifically attached to areas outside the patterns. Cells were vitrified 4-6 h post-transfer for RPE cells (to attain a higher number of individual grid squares with a single cell) or after overnight incubation for HeLa cells.
Live Cell Confocal Imaging
Time lapse imaging of HeLa cells on grids (
Zeiss Airyscan microscopy
AiryScan microscopy of RPE cells on patterned grids (
Widefield Microscopy Imaging
Epifluorescence images (
Vitrification
Grids were blotted from the reverse and immediately plunged into a liquid ethane or ethane/propane mixture at liquid nitrogen temperature using a Leica EM GP plunger (Leica Microsystems, Vienna, Austria). The plunger was set to 37° C., 99% humidity, and blot time of 2 s for R2/1, and 2.5 s for R1/4 and R1/20 grids. The frozen grids were stored in sealed boxes in liquid nitrogen until further processing.
Cryo-Scanning Electron Microscopy and Focused Ion Beam Milling
Cryo-FIB lamella preparations were performed as described in Ref. 5, on a dedicated dual-beam microscope with a cryo-transfer system and a cryo-stage (Aquilos, ThermoFisher Scientific, Brno, Czech Republic). Plunge frozen grids were fixed into autogrids modified for FIB preparation (Max Planck Institute of Biochemistry, Martinsried, Germany), mounted into a shuttle (ThermoFisher Scientific) and transferred into the dual-beam microscope through a load-lock system. During FIB operation, samples were kept at constant liquid nitrogen temperature using an open nitrogen-circuit, 360° rotatable cryo-stage. To improve sample conductivity and reduce curtaining artifacts during FIB milling, the samples were first sputter-coated with platinum (10 mA, 20 s) and then coated with organometallic platinum using the in situ gas injection system (GIS, ThermoFisher Scientific, Netherlands) operated at room temperature, 10.6 mm stage working distance and 7 s gas injection time. Appropriate positions for FIB preparations were identified and recorded in the MAPS 3.3 software (ThermoFisher Scientific, Brno, Czech republic), and eucentric height refined per position. Lamellae or wedges were prepared using Gallium ion beam at 30 kV at stage tilt angles of 20° for lamellae and 12°-13° for wedges. Lamella or wedge preparations were conducted in a stepwise rough milling, starting with high currents of 1 nA, 5 □m away from the area of interest, gradually reduced to lower currents, down to 50 pA for the final cleaning steps. Progress of the milling process was monitored using the scanning electron beam operated at 10 kV and 50 pA (or 2 kV for visualization of micropatterns). For improved conductivity of the final lamella for specimens intended for phase plate tomography, we again sputter coated the grid after cryo-FIB preparation with platinum (10 mA, 3 s). Grids were stored in sealed boxes in liquid nitrogen until further processing.
Cryo-Electron Tomography
Cryo-electron microscopy data were collected on a Titan Krios microscope operated at 300 kV (ThermoFisher Scientific, Netherlands) equipped with a field-emission gun, a Quantum post-column energy filter (Gatan, Pleasanton, Calif., USA), a K2 Summit direct detector camera (Gatan) and a Volta phase plate (ThermoFisher Scientific, Netherlands). Data were recorded in dose-fractionation mode using acquisition procedures in SerialEM software v3.7.2Ref. 21. Prior to the acquisition of tilt-series, montages of the entire lamella were acquired at ˜2 nm/pix. Tilt-series using a dose symmetric scheme were collected in nano-probe mode, EFTEM magnification 42,000× corresponding to pixel size at the specimen level of 3.37 Å, 3-4 μm defocus, tilt increment 2° with constant dose for all tilts, total dose ˜120 e−/Å2. The pre-tilt of lamellae with respect to the grid plane due to cryo-FIB milling at shallow angles (10-15°) was corrected for by tilting the stage on the microscope. Conventional tilt-series, Volta phase plate (VPP), were acquired at the same settings with an objective aperture and a beam tilt of 4 mrad for autofocusing (tomograms in
Data Processing
Prior to tilt-series alignment, the projection movies were corrected for beam induced drift in the SerialEM plugin. Tilt series alignment and tomographic reconstructions were performed using the IMOD software package, version 4.9.0Ref. 21. In absence of fiducial gold nanoparticles in the FIB-lamellae, alignment of tilt-series images was performed with patch-tracking. Final alignment of the tilt-series images was performed using the linear interpolation option in IMOD without CTF correction. Aligned images were binned to the final pixel size of 13.48 Å. For tomographic reconstruction, the radial filter options were left at their default values (cut off, 0.35; fall off, 0.05). Tomograms from
Photo-Micropatterning by a 355 Nanoablation UV-Laser
The inventors used a second method of photo-micropatterning by ablating the PLL-g-PEG passivation layer in a spatially-control manner using a 355 nm-pulse laser setup (see methods). Due to the pulsing nature of the laser, it has to scan the region of interest to be patterned to ablate the anti-fouling agent. The action of the laser leaves an impression on the film that is visible by light microscopy (
The inventors developed a method for producing a circuit of cells, comprising the functionalized electron microscopy support according to this invention, and at least two cells. The circuit of cells can, for example, grow on a device, such as a chip. Said cells can be neurons, hepatocytes, myocytes, cardiomyocytes, stem cells, stem cell progenitor cells, trophoblasts, astrocytes, glial cells, enterocytes, hepatic cells, kidney cells, endothelial cells, epithelial cells, such as biliary epithelial cells, syncytiotrophoblasts, cytotrophoblasts, mesenchymal cells, inner cochlea cells, outer cochlea cells, and/or trophoblasts. As a proof of principle, the inventors developed a human neural network growing on a functionalized electron microscopy support according to this invention (
The micropattern was coated with laminin protein for human neuron adherence, and grids were seeded with induced human stem cells and differentiated to neurons (day-6 post seeding) on a grid. Human neurons can be observed by fluorescence microscopy due to a soluble protein (Ngn2-GFP) spread across the cells. Neurons are observed following the micropatterned circuit (
The references are:
| Number | Date | Country | Kind |
|---|---|---|---|
| 19181388.0 | Jun 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/067189 | 6/19/2020 | WO |
