Prepare 1 mg/ml solution in Hepes 10 mM pH 7.4 as recommended by the manufacturer. Aliquot a 100 µl volume (or a convenient amount), which would serve to treat ~20 grids, and store at -20°. Dilute the solution in the same buffer to 0.5 mg/ml immediately before use.
Note: PLL-g-PEG can be stored for one week at 4°. Nevertheless, we recommend to use it immediately after its preparation or thawing.
Use low-pressure plasma cleaning to oxidize the grids and render them hydrophilic.
o Place grids onto a glass slide and plasma clean both sides. For SiO2 film use a 100W power with a 10 cm3/min flow rate of oxygen gas for 30-40 s (each side), while for carbon films a 30W power for 10-30 s should be used.
Note: we have also employed successfully different equipment for this step, e.g., plasma cleaning under atmospheric air at 30W for 45 sec at 0.039 mbar (i.e., Pelco EasyGlow Glow Discharge Unit. Ted Pella, Redding, USA, Cat. no. 91000) for any grid, or 0.03 mbar (Diener Zepto Plasma, Diener electronic, Ebhausen, Germany) using a 100W for SiO2 and 30W for carbon films for 30 s. We additionally tested a mixture of oxygen/argon 75/25% (Nano Clean Plasma cleaner. Fischione Instruments, Hanau, Germany) at 100W for SiO2 and 30W for carbon films for 30 s.
o Prepare a humid chamber: 100 mm culture dish, containing water-soaked filter paper and a parafilm on top.
o Place 20 µl droplets of PLL-g-PEG (0.5 mg/ml) on the parafilm. Each droplet is used to passivate a single grid.
o Place the plasma cleaned grids on the PLL-g-PEG droplets, and incubate in the humid chamber for 1h at room temperature or overnight at 4°C (sealed with parafilm).
CRITICAL: do not let the PLL-g-PEG dry out on the grid during the passivation.
Alternative two-step passivation:
o Incubate the grids on 20 µl droplets of 0.01 % PLL overnight on parafilm in a humid chamber at room temperature.
o Wash the grids five times in 100 mM NaHCO3 pH 8.4, and incubate for 1-2 h with 50 mg/ml PEG-sva dissolved in the same solution.
o Wash the grids five times in 100 mM NaHCO3 pH 8.4.
Note: the two-step passivation is a convenient alternative in the absence of a plasma cleaner.
Design of micropatterns
The design of micropatterns depends on the microscope set-up used. For example, if nanoablation with a 355 nm pulsing laser is performed, the micropattern design will be limited by the features and performance of the confocal microscope software. If using the PRIMO module (Alvéole Lab, Paris, France), patterns can be designed in Inkscape (http://www.inkscape.org/) as 8-bit binary files and exported as png files according to the PRIMO manufacturer’s instructions, which can be loaded into the Leonardo software v4.12 (Alvéole Lab, Paris, France).
Note: when designing the patterns in Inskape, it is important to consider the calibration of the equipment, i.e., pixel to µm ratio, as well as the size of the DMD mirror, for proper sizing of the pattern and the estimated number of expositions. These parameters will dictate the patterning time.
Micropatterning of grids (two methods)
1. 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, a UPLSAPO 63x (NA 1.35) oil objective, and a standard PMT or GaAsP PMT detectors.
The 355 nm laser had an average power of 50 mW, 500 ps pulse width, 1kHz repetition rate, and a maximum energy per pulse of 30 µJ.
o After passivation with PLL-g-PEG, blot-dry the grid from the back on a Kimtech wipe, and quickly place the grid with the SiO2 film facing down (towards the objective) on a 1-3 µl drop of either Hepes 10 mM pH 7.4 or PBS in a sealed glass bottom low 35 mm ibidi µ-Dish.
Note: high humidity should be kept using water-soaked filter paper inside the dish to avoid buffer evaporation.
Note: only grids with SiO2 films were micropatterned with this technique. For carbon and gold films, the laser conditions need to be adjusted, and especially tuned to a lower power for carbon.
o Use transmission and/or reflection (e.g., 488 nm laser) to focus on the grid film.
o Draw patterns (ROIs) of square or circular shape (between 10 to 40 µm diameter, depending on the aim of the experiment) using the Olympus FV 10-ASW software.
o Focus on the film and generate micropatterns by applying a 10-11 % laser power, 40 μs per pixel and 10 iterations.
o Move to the next grid square, focus on the film, and apply the laser. Repeat this step until the desired area is covered (e.g., 6x6 grid squares at the central area of the grid).
2. PRIMO™ (DMD-based illumination + Photo-activator):
Micropatterning is performed using a 375 nm laser (4.5 mW) and a digital mirror device (DMD) to generate a spatially controlled laser illumination of the sample (PRIMO™, Alvéole Lab, Paris, France). Resolution limit: ~1.2 µm. In this case, an inverted Nikon microscope Ti-E equipped with a CFI Super Plan FLuor 20x 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 (Alvéole Lab, Paris, France) was used.
o After passivation with PLL-g-PEG, blot-dry the grids from the back on a Kimtech wipe, and quickly place it (with the film facing towards 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.
CRITICAL: do not let the PLPP solution dry out on the grid.
Note: high humidity should be kept using water-soaked filter paper inside the dish to avoid PLPP evaporation.
Note: the microscope stage and photo-patterning were controlled with the µmanager software by the Leonardo plugin software.
Note: although PLPP solution is stable (as per manufacturers), it is recommended to aliquot a convenient volume and freeze at -20°.
o Load the png file with the design of the micropattern in the Leonardo software, and accommodate (rotate and translate) the pattern to the grid position. Select the stitching mode, and use a final dose of 800‑1000 mJ/mm2 (equivalent to ~30 s per DMD exposition).
Note: continuous focusing on the film is required during the patterning period. The automated hardware-based focus system of the microscope employed here is unable to accurately and reliably focus on the grid film. Therefore, on the fly continuous and manual focusing on the film is required while the automated patterning is running. The focusing step will highly depend on the quality and flatness of the grid. Focusing could be further optimized by generating a tool (e.g., script) to keep the focus on the grid film while patterning.
o Retrieve the grid from the PLPP solution and wash in a 1000 µl drop of water, followed by two consecutive washes in 300 µl drops of PBS.
Pause point: at this point, one-step passivated and micropatterned grids can be stored in PBS at 4°C in a humid chamber, remaining functional for at least 30 days. Alternatively, grids can be stored dried, after the water washing step, remaining functional for up to ~7 days in atmospheric conditions. However, although grids were functional after 1 h – overnight rehydration, their performance appears superior when stored wet.
General remarks and guidelines for micropatterning:
Minimize the time that the grids are kept in the PLPP solution. Grids were typically retrieved from the PLPP solution immediately after patterning, washed in abundant water and placed into the corresponding buffer for the next step.
Patterning of 4 grids (using the Primo module) with a 6 x 6 or 8 x 8 grid squares area will take approximately 30 min or 1 h, respectively. To pattern 4 grids, 40 µl of 1 mg/ml PLL-g-PEG is required, and takes a minimal time of 3 hours, including grid functionalization.
Optional: a silicon stencil can be used to keep the grid on place11.
o Incubate grids in a 20 µl drop of either 50 µg/ml fibronectin (prepared in PBS), or 30 µg/ml of fibrinogen-Alexa546 (prepared in 100 mM NaHCO3 pH 8.4) on parafilm for 1h at room temperature.
Note: fibrinogen-Alexa546 is used to test for proper micropatterning, while fibronectin is meant for cell adhesion. A combination of both can also be used in a 1:5 or 1:10 ratio (fibrinogen:fibronectin).
o Wash the grids three times in 300 µl drops of the corresponding buffer (PBS for fibronectin and, 100 mM NaHCO3 pH 8.4 for fibrinogen).
Pause point: fibronectin‑treated grids can be stored in PBS in a humid chamber at 4°C, remaining functional for at least 10 days. The maximum active life time of protein‑functionalized grids remains unknown. This will depend on the protein stability itself.
o Check for micropatterned areas by fluorescence microscopy (detection of Alexa546) if applicable.
o Retinal pigment epithelium (RPE1) cells were cultured in 5 ml of DMEM F-12 + GlutaMAX, and HeLa cell lines in DMEM + GlutaMAX, in 50 ml culture flasks. Both were supplemented with 100 mg/ml of penicillin and streptomycin. Incubate the cultures at 37° with 5% CO2.
Note: Cells should ideally be at maximum 60% confluency.
o Transfer the grids (film facing upwards) to a 35 mm ibidi dish high, filled with 1 ml of medium.
o Detach cells from flask by trypsinization using 2 ml of Trypsin-EDTA (0.05%), retrieve the solution and incubate for 2 min at 37°. Check under the microscope for cells rounding, and resuspend cells in 1 ml of medium.
o Count cells by mixing 10 µl of TC10 Trypan Blue Dye with 10 µl of the cell suspension. Mix by pipetting up and down gently 20 times.
o Pass the cells trough a strainer and prepare 80,000 RPE1 cells or 200,000 HeLa cells in 1 ml of medium. Add the cells to the 35 mm ibidi dish with the grids, equivalent to a density of 8×103 cells/cm2 and 2×104 cells/cm2, respectively.
CRITICAL: perform the cell strainer step immediately before seeding.
o Incubate the cells on the grids for 20-35 min for RPE1 cells or 1.5-2 h for HeLa cells, to allow initial attachment.
Note: cell number and timing of seeding needs to be optimized for each cell type.
o Transfer the grids to a new cell-free dish with medium. Briefly, check grids for seeding by transmitted light microscopy (e.g., Olympus CKX41 widefield microscope equipped with the CellSens Entry 1.9 software).
Note: the transfer to a new dish is beneficial to avoid attachment of multiple cells per pattern, and also to avoid eventual non-specific attachment of cells on non-patterned areas.
o Incubate the dish at 37°C with 5 % CO2 to allow full cell adhesion to the grids.
Note: Seeding should be performed in a sterile laminar flow hood.
Vitrification, Cryo-FIB milling and Cryo-ET
o Vitrify cells after 4-6 h post-transfer for RPE1 cells (to attain a higher number of grid squares with a single cell) or after overnight incubation for HeLa cells.
o Cells can be plunge-frozen in a Leica GP EM. Settings: 1.5 s blotting for R2/1 and R1/4 grids, and 2.5 s blotting for R1.2/20 holey and continuous film. For further details of vitrification, cryo-FIB milling, and cryo-ET please refer to5,10.
o Grids should be stored in sealed boxes in liquid nitrogen until use.
All micropatterning steps and grid treatments were performed under sterile conditions using a Bunsen burner. Grids were handled with a tweezer n° 55. FluoroBrite™ medium was used for live cell imaging.