Proper preparation ensures reliable probe performance and consistent injection results. This section covers how to prepare FluidFM probes using plasma treatment and antifouling coatings, optimize cell culture conditions for injection workflows, and configure washing plates to address common issues such as air bubbles and debris.
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 800 | Cytosurge)
- Plasma surface treatment machine (e.g., Diener Zepto One, Harrick Plasma Basic plasma cleaner)
Procedure
- Place the probe with the blister pack into a plasma cleaner (remove the cover first).
- If the blister pack doesn't fit into the plasma cleaner, remove the probe and place it on a glass slide with the cantilever facing upward, then place the slide into the plasma cleaner.
- 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:
- Generator frequency: 100 kHz.
- Power: 30 W.
- 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: Pure O₂ plasma is more aggressive and may damage probe adhesives; therefore, it is not recommended. If O₂ must be used, reduce treatment time to 10–15 seconds as a preliminary test. Synthetic air (N₂/O₂ mixture) at standard composition should produce comparable results to atmospheric air, though this has not been validated in our laboratory.
Probe Coating
To avoid the adhesion of cell debris or deposition of the sample around the aperture of the cantilever, the FluidFM Nanosyringe requires coating. This thin layer acts as an antifouling agent, preventing the accumulation of biomolecules and cell debris on the cantilever during injection.
Probe coating with Sigmacote®
Sigmacote® is a silane solution in heptane that increases hydrophobicity and improves antifouling properties on the external cantilever surface.
Materials
- Plasma treated FluidFM Nanosyringe Precision Plus 800 (see previous section)
- FluidFM Coating kit: desiccator with desiccant cartridge, connected to a vacuum tabletop pump
- Disposable wipe (e.g., kim-wipe)
- Sigmacote® (Merck, Cat.No. SL2 – 100ML)
Warning! When handling Sigmacote®, please note the following:
- Handling of Sigmacote® requires safety precautions. Refer to the Merck Sigmacote® Safety Data Sheet (SDS), document 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.
Procedure
- Prepare the desiccator with one disposable wipe, a desiccator cartridge and a container for the probe (e.g., 12-well plate).
- Place the probe in the
container inside the desiccator and add 1 mL of Sigmacote® on the disposable wipe. Close
the desiccator immediately after.
Depositing the Sigmacote® in the disposable
wipe inside the desiccator chamber, together with the desiccant cartridge and
the FluidFM probe.
- Apply continuous vacuum for 1 hour to coat the probes. Then, carefully release the pressure from the desiccator.
- Place the probes in an oven at 60°C for 1 hour to ensure optimal coating.
- Coated probes are ready for use and remain effective for at least 5 days.
Probe coating with PAcrAm
PAcrAm is a polymer used as antifouling agent for glass and polymer cell culture substrates. Although it is primarily recommended for pick-and-place workflows, it becomes helpful for injection experiments when injection mix adheres to the surface of the microchannel or when the chosen cell line adheres to the cantilever even with Sigmacote® (e.g. cardiomyocites or HEK293). PAcrAm is distributed in 0.1 mg lyophilized aliquots, which also contain the buffer required for surface modification.
Materials
- Isolation coating aliquot
- Ultrapure water
- PBS
- Injection mix [Insert link to section]
- 12-well plate (Thermo Fisher Nunc, Cat. No. 150628)
Procedure
The coating of the probes with PAcrAm takes place inside the FluidFM OMNIUM at 37°C. Make sure to warm up the system before the coating starts.
- Equilibrate the PAcrAm aliquot to room temperature. Dilute the content in 1 mL of ultra-pure water (pre-warmth at 37°C) . Vortex for 5 seconds.
- In a 12-well plate, dispense all the PAcrAm solution into one of the wells. Prepare two additional wells with 1 mL of ultrapure water and three wells with 1 mL of PBS each. Use the Exchange Plates workflow in ARYA to place the coating plate on the left port.
- Fill the reservoir of the FluidFM probe with 1 μL of the injection mix (IM).
- Select Preparation Advanced workflow and follow it until the step Go to Sample. In this step, right after filling the probe, dip the FluidFM probe into the PAcrAm solution on the plate placed in the left port, using the Navigation Tool.
- Apply −250 mbar pressure for 5–10 seconds. This step ensures the internal microchannel surface is also coated.
- Incubate the probe in PAcrAm solution for 30 minutes while applying −20 mbar pressure to the cantilever.
- Use the Washing Tool to wash the probe in two wells of ultrapure water and three wells of PBS. Finish the Preparation Advanced workflow. The probe is now ready to use.
Cell culture preparation
The FluidFM OMNIUM system supports a wide range of plates and dishes. 24-, 12- and 6-well plates are compatible, as are standard dish sizes of various formats. To avoid collisions with system components, use only plates that have been tested for compatibility (see a list of recommended plates and dishes below).
Each new plate or dish has a specific configuration in the system. With the plate editor, integrated in the ARYA software, a template can be created for each case. Contact Cytosurge for assistance with creating new templates . Cytosurge can create a validated configuration for custom containers upon request.
Custom plate holders can be fabricated by the user or ordered from Cytosurge (subject to charges). Examples include holders designed for Willco dishes and microscopy glass slides.
Use the correct volume of medium or aqueous solution in each well: Excessive volumes can produce background noise during imaging; insufficient volumes can result in poor laser signal quality and approach errors. The table below lists recommended volumes for each plate type:
Plate/Dish | Company | Catalog Number | Recommended volume |
6-well cell culture plate | Costar (Corning) | 2 mL | |
12-well cell culture plate | Costar (Corning) | 1 mL | |
12-well cell culture plate | Thermo Fisher Nunc | 1 mL | |
24-well cell culture plate | Costar (Corning) | 500 μL | |
Ibidi dishes, 35 mm | Ibidi | 2 mL | |
WillCo dishes, 50 mm | Willco wells | 4 mL |
Regarding the cell culture, cell media composition and seeding density should be optimized based on experimental goals and system configuration:
- If the OMNIUM system is not equipped with CO₂ control, use CO₂-independent media during the experiment. To reduce background fluorescence during imaging, use phenol-red free media.
- The optimal cell density depends on the cell type and experimental objectives (e.g., for long-term tracking experiments, account for cell doubling time to prevent confluency during observation). Lower densities also facilitate easier cell tracking post-injection. Finally, cell debris or excessive floating cells can interfere with the laser signal: ensure cells are well-adhered before beginning the experiment (e.g., by seeding them the day prior to the experiment).
Washing Plate
The injection workflow requires different wash sequences depending on the situation:
- When an air bubble interferes with the laser signal, perform an ethanol wash.
- When cell debris remains in the cantilever after injection, perform a Terg-a-zyme wash.
The wash plate configuration is as shown below.
Wash plate configuration showing well positions for distilled water, 70% ethanol, and 1% Terg-a-zyme solutions.
In the wash tool in ARYA, create the following sequences:
Ethanol Wash
Use this wash sequence if the probe has an air bubble after entering liquid. The wash sequence for an ethanol (bubble) wash is as follows:
- A2: Keep pressure for 3 seconds
- B2: Keep pressure for 3 seconds
- B1: Keep pressure for 1 second
- C3: Keep pressure for 3 seconds
- A1: Keep pressure for 3 seconds
Terg-a-zyme Wash
Use this wash sequence if the probe has visible debris on the tip or if a cell ruptured during injection. The wash sequence for a debris wash is as follows:
- A2: 800 mbar for 5 seconds
- B2: 200 mbar for 5 seconds
- B3: 200 mbar for 30 seconds
- C3: 200 mbar for 5 seconds
- A1: 200 mbar for 5 seconds
Important: Refresh wash solutions every 4–5 hours during experiments, or when contamination/debris is visible, for optimal performance.