Conducting a feasibility analysis requires experimental preparation:

• Probe coating
• • Plasma treatment
  • Sigmacote® coating
  • PAcrAm coating
• Biopsy plate preparation
• • Cell preparation
  • Drying area preparation
  • Droplet handling area preparation
• Wash plate preparation

Probe Coating

All probes that are used for cell biopsy should be coated with Sigmacote® (Merck) both inside and outside the cantilever. Sigmacote® is both antifouling, preventing biomolecules from sticking to the probe surface, and hydrophobic, allowing oil to run smoothly inside the cantilever. Additionally, we recommend a second coating with PAcrAm-g-(PMOXA, NH2, PFPA) outside the cantilever. PAcrAm is a polymer that reduces the air bubbles occurrence and eases the removal of oil spillages, showing also stable oil-extract fronts. For biopsy experiments, we recommend that the FluidFM Nanosyringe (BIO FluidFM Nanosyringe Precision Plus 550 | Cytosurge) is used. Probes should be coated 1-5 days before use. To coat the probes, first plasma clean them.

Plasma Treatment of FluidFM Probes

Each FluidFM Nanosyringe should be plasma cleaned prior to the experiment. The plasma treatment ensures a clean probe surface for coating, as it removes organic contaminants. It also introduces functional groups, resulting in a hydrophilic surface that promotes effective coating. This can significantly improve the quality of any coating and make filling of the probe faster and easier.

Materials

• FluidFM Nanosyringe (BIO FluidFM Nanosyringe Precision Plus 550 | Cytosurge)
• Plasma surface treatment machine (e.g., Diener Zepto One, Harrick Plasma Basic plasma cleaner)

Procedure

1. Place the probe with the blister pack into a plasma cleaner (remove the cover first)
2. 1. If the blister pack doesn't fit into the plasma cleaner, take the probe out and put it on top of a glass slide with the cantilever upwards into the plasma cleaner.
3. Plasma clean the FluidFM probe(s) using low-pressure (vacuum) plasma cleaning with air as the process gas for 2 minutes using the following parameters: 
4. 1. 100 kHz frequency
   2. 30W power
5. Immediately start the coating treatment after plasma treatment (at the latest, 10 minutes after plasma cleaning).

Important: Longer plasma cleaning times can have a negative impact on the probe glue and should be avoided

Notes for alternative process gases: If using pure O2, consider reducing the treatment time (starting with ~10-15 seconds as a preliminary test), as oxygen plasma is more aggressive and may potentially damage adhesives at standard treatment time, and therefore is not recommended. If using synthetic air (N2/O2 mixture); standard composition should produce comparable results to atmospheric air, though this has not been validated in our laboratory.

Following plasma cleaning, coat the probes using Sigmacote® and the coating kit.

​​FluidFM Probe Coating with Sigmacote® Inside the Channel

The FluidFM Nanosyringe is coated to increase hydrophobicity and improve anti-fouling properties outside and inside of the cantilever. This is specifically relevant for the single cell biopsy application where the cantilever is filled with mineral oil and the cellular content is frontloaded multiple times in the cantilever.

Materials

• Coating container CYPR/003407 (see Figure 1)
• Sigmacote® (Cat. No. SL2-100ML; Sigma Aldrich)
• FluidFM Nanosyringe (Cytosurge/BIO FluidFM Nanosyringe Prescision Plus 550: BIO FluidFM Nanosyringe Precision Plus 550 | Cytosurge) that has already been plasma cleaned (see Plasma Treatment of FluidFM Probes)
• FluidFM Coating kit 

 Warning! When handling Sigmacote®, please note the following:

• Handling of Sigmacote® requires safety precautions. Please see (Merck/Sigmacote/SDS: SL2)
• Sigmacote® is sensitive to water. Avoid condensation by equilibrating the solution to room temperature prior to use.
• The effectiveness of Sigmacote® decreases with age. Only use Sigmacote® that has been open for less than three months. To help prolong the lifetime, consider aliquoting a new bottle after opening for the first time.

Figure 1. Coating container. 

Procedure

1. There are two rubber o-rings for each probe chamber, one on the container and one on the lid (see Figure 1). Make sure that they are fully tucked into the trench and are not bulging out.
2. Add 5 µL of Sigmacote® to the bottom of each chamber, and place the FluidFM Nanosyringe in the corresponding chamber.
3. Align the lid with the container. The chambers are numbered for easier probe tracking.
4. Close the container by gently screwing the lid. Do not overtighten, it can cause breakage!
5. Attach the tubing to the corresponding outlet – each probe chamber has its own outlet. For coating more than one probe at a time, the individual tubing of the chambers should be connected (see Figure 2).
6. Connect the longest tube to the vacuum pump.
7. Turn on the vacuum pump and leave for 1h. Confirm that the manometer reaches at least -700 mbar.
8. After coating is finished, carefully release the pressure before taking out the FluidFM Nanosyringe.
9. Remove the FluidFM Nanosyringe from the coating container and place it in the oven for post-treatment for 1h at 60 °C.
10. • Note: We recommend using a manometer on the vacuum pump and monitoring negative pressure. It should reach below –700 mbar.

Figure 2. Schematic of the coating connector. Note the different tubing configurations depending on the number of probes to be simultaneously coated. 

Cleaning of the coating containers

Sigmacote® coating containers are made from resin, and continuous use can cause the formation of particles, which should be removed prior to the coating procedure to avoid clogging of the FluidFM Nanosyringe. The containers can be reused by following a simple wash protocol:

1. Immerse the containers in isopropanol (technical grade is sufficient).
2. Sonicate for 5 minutes at room temperature.
3. Pour out the used isopropanol and repeat sonication once.
4. Rinse with isopropanol to remove all the loose particles.
5. Dry the container with compressed air.
6. Store the container closed at room temperature or use it straight away for coating.

Coating Troubleshooting

If the vacuum cannot be properly reached, consider checking whether the O-rings are properly housed and all tubes are properly connected. Insufficient coating leads to​ poor (re)usability of the FluidFM Nanosyringe probes, and​ inability to release the extract.​ Excessive coating leads to​ unfillable probes. If coating issues keep persisting, reach out to Cytosurge (support@cytosurge.com) for troubleshooting advice.

FluidFM Probe Coating with PAcrAm Outside Channel

The FluidFM Nanosyringe should be additionally coated with PAcrAm-g-(PMOXA, NH2, PFPA) on the outside of the cantilever after Sigmacote® coating. PAcrAm coating reduces air bubble formation during oil handling and facilitates the removal of oil spillages, while also stabilizing the oil-extract interface. This coating is specifically beneficial for single-cell biopsy applications involving multiple preloading cycles.

Materials

• PAcrAm-g-(PMOXA, NH2, PFPA) (Cytosurge/Extraction coating aliquot).
• FluidFM Nanosyringe coated with Sigmacote (see FluidFM Probe Coating with Sigmacote® Inside the Channel)
• Mineral oil (Cat. No. M8410-5ML (Merck/Mineral oil: Mineral oil light oil neat, BioReagent, mouse embryo cell culture 8042-47-5))
• 50mM Na2CO3 buffer, pH 10 (in UltraPure - RNase-free water), filtered and warmed up (37°C).
• 2-propanol
• UltraPure - RNase-free water
• 12-well plate (Thermo-Fischer, Nunc; cat. Num. 150628)
• FluidFM OMNIUM system
• FluidFM Probe Loading plate
• Table-top centrifuge

Procedure

1. Prepare the PAcrAm solution by dissolving one aliquot of PAcrAM-g-(PMOXA, NH2, PFPA) in 1 mL of warm 50 mM Na2CO3 buffer, pH10 (final concentration: 0.1 mg/ml). Resuspend by pipetting.
2. Fill the FluidFM Nanosyringe reservoir with 1 μL of sterile-filtered mineral oil (0.22µm filter).
3. 1. Ensure the pipette tip does not touch the sides of the reservoir to avoid leaving oil on the prism of the head. If oil is present on the sides, use a wet wipe (ethanol) to carefully remove it.
4. Place the nanosyringe in the probe loading plate inside the OMNIUM instrument and follow the Preparation Advanced workflow
5. 1. Measure the spring constant and record the value for later use.
   2. Note: When filling the probe, use low pressure (300–400 mbar). Stop filling immediately when the oil reaches the tip of the cantilever to prevent oil from exiting and accumulating on the tip. This prevents the oil exiting the cantilever and accumulating on the tip.
6. Stop the Preparation Advanced workflow once filled and proceed to PAcrAm coating steps.
7. Load the washing plate into the OMNIUM:Figure 3. Wash plate configuration for PAcrAm Coating. 
8. Set the system pressure to 100 mbar before cleaning to prevent 2-propanol from entering the probe channel.
9. Clean the outside of the probe by dipping into the three 2-propanol wells sequentially (3 seconds each, 100 mbar).
10. Dip the probe into the Na2CO3 well (5 sec, 1 mbar) at idle pressure of 1 mbar
11. 1. Note: If air bubbles form, re-enter the probe into the Na2CO3 well to remove them. Do not return to IPA
12. Transfer the probe to the PAcrAm well and lower it to content height to fully submerge the cantilever.
13. 1. Note: The washing tool does not automatically lower the probe to content height. Manually adjust the z-position or use the navigation tool to position the probe in the well.
14. Incubate the probe in the PAcrAm solution at room temperature for at least 1 hour (0 mbar).
15. Rinse the probe by dipping into three consecutive wells of ultrapure RNase-free water (1 mL/well).
16. 1. Note: at this point, the nanosyringe can be stored in a water well for up to 1 day or dried (by air drying or using the Probe Drying procedure defined in the Experimental Procedure section, in Buffer testing - probe drying) and stored in the dark for up to several months.
17. In ARYA, return to the Preparation Advanced workflow and complete the remaining steps:
18. 1. Measure the sensitivity in cell-free well containing liquid.
    2. When aligning the probe, use 10× or 20× magnification (choose based on your imaging setup). Proper probe alignment is crucial for volume estimation tool and cell targeting.
    3. Dry the cantilever on the drying pad. Proceed only once the cantilever is fully dry.
19. The nanosyringe is ready to use. 
20. 1. The coated nanosyringe can be kept in a water well until usage for at least 1 day or dried (by air drying or by drying the probe in the FluidFM OMNIUM) and stored in the dark for up to several months. 

Biopsy Plate Preparation

The single cell biopsy plate is designed to support the single cell biopsy workflow and enable the user to obtain subcellular extracts with the FluidFM OMNIUM system. It should be set up as described below before the biopsy experiment. The biopsy plate is shown in Figure 4.

Figure 4. The single cell biopsy plate and its features.

The components of the biopsy plate include:

• Ibidi dish holder
• Ibidi µ-dish 35 mm, high
• Whatman grade 3 qualitative filter papers ø 23 mm
• Drying paper holder
• Droplet slide
• Droplet slide holder

Cell Handling Area

Cells have a dedicated place on the biopsy plate that fits a standard or a gridded 35 mm ibidi dish (Cat. No. 81148 (µ-Dish 35 mm, high Grid-50 Glass Bottom | ibidi), Cat. No. 81166 (µ-Dish 35 mm, high Grid-500 | Gridded Bottom Dish | ibidi) or Cat. No. 81156 (µ-Dish 35 mm, high | Cell Culture Imaging Dish | ibidi)). We recommend using gridded dishes as they enable better traceability of cells. The ibidi dish holder is designed specifically to tightly hold an ibidi dish and minimise rotation, and it should be exchanged if the dish is no longer held tightly. To prepare the cells for experimentation:

1. Clean and coat the ibidi dish if required (according to the cells’ requirements).
2. Seed cells in the ibidi dish at a concentration that was defined in the Cell Line Onboarding. (Seeding Density and Timing Tracking-Interval). 
3. Fit the ibidi dish into the ibidi dish holder. Make sure that the plate is pushed to the bottom of the holder evenly.
4. Place the ibidi dish holder with the dish in the biopsy plate.
5. Remove the lid of the ibidi dish after placing the plate in the OMNIUM.

Drying Area​

Within the biopsy workflow, the probe is wetted during and after the cytoplasm extraction but needs to be dry to be able to enter a droplet where the preload will be collected or where the biopsy will be deposited. A dry probe is essential for:

• Successful droplet entry.​
• Avoiding contamination of deposition buffer.​

Whatman grade 3 qualitative filter paper is used to dry the probes. Drying requires the user to cut the Whatman paper in half and insert the halves in the holder, creating a slit where the probe will be dried. A specifically designed paper cutting guide at the back of the paper holder is used to cut the paper in equal halves. To prepare the drying area for experimentation:

1. Take 3 × Whatman grade 3 qualitative papers
2. Cut the three filter papers in half by using the guide on the back of the drying station and a sterile scissors (see Figure 5).
3. Place each paper half into the designated area on the plastic holder (see Figure 5). Two halves of the paper are placed per one drying spot. Like this, a slit is created for drying, which allows the consumable to touch the paper, resulting in a dry cantilever. Make sure that the cut halves are placed securely and straight to the Whatman paper holder, as poorly placed papers can result in disengagement of the Z-stage during automatic drying.
4. Place the plastic holder in the drying area of the biopsy plate.

Figure 5. Whatman paper holder. Left: a view of both sides of the holder. Right, how to use the paper cutting guide.

Droplet Handling Area

The droplet slide is a reusable glass slide with hydrophobic coating. The slide has 30 unique droplet positions defined with circular marks, which enables fast and precise positioning of the buffer droplet. Biopsies are deposited in a 1 µL droplet, so the small content of RNA stays as concentrated as possible. The droplet slide holder stabilizes the droplet slide position since the slide remains inside the holder when placing the droplets. The droplet slide should be cleaned prior to each experiment to avoid cross-contamination between the experiments. The cleaning protocol will depend on the whether or not the experiment requires a clean collection. For all experiments, wipe the droplet slide with 70% ethanol, followed by water. Additionally, for clean experiments, then UV both sides of the slide for 30 minutes each. To prepare the droplet handling area for experimentation:

1. Clean the droplet slide.
2. Place the droplet slide in the droplet slide holder.
3. 1. Note: A1 needs to be in the upper left corner.
4. Place the droplet slide holder in the droplet handling area of the biopsy plate.

Wash Plate Preparation

The biopsy workflow requires specific wash sequences at various points in the biopsy process. Some wash sequences are only used as required, while others are carried out after each biopsy.

The wash plate configuration for biopsy experiments is as shown in Figure 6 below.

Figure 6. Wash plate configuration for cell biopsy. 

The wash sequence for an ethanol (bubble) wash is as follows:

1. A2: Keep pressure for 3 s
2. B2: Keep pressure for 3 s
3. B4: Keep pressure for 1 s
4. B3: Keep pressure for 3 s
5. C2: Keep pressure for 3 s

Use this wash only when the probe has an air bubble surrounding it after entering a well of liquid.

The wash sequence for a water wash is as follows:

1. A1: Keep pressure for 3 s
2. B1: Keep pressure for 3 s
3. C1: Keep pressure for 3 s

Use this wash after each biopsy, before drying the probe for the extract deposition.

The wash sequence for a blocked probe or aspirated media is as follows:

1. C3: Alternate pressure between high and low so that the probe empties and is rinsed several times.
2. If this does not work, repeat in well A4.
3. Once the probe is working and empty, use the water wash sequence to rinse the Triton-X from the probe.

Refresh wash plate solutions after half a day of experimentation or after approximately seven biopsies.