3D Printed PDMS Chambers

Introduction

Highly detailed imaging chambers can be constructed by 3D printing a mould using DLP (resin) printing, which is then used to cast a PDMS chamber that can be plasma-fused to a glass coverslip appropriate for high-resolution microscopy (e.g. #1.5 thickness). The resolution of features in these moulds is determined by the printer, with our current SLA printer (Elegoo Mars Pro) providing an X/Y resolution of 47 μm and a z-resolution of 50 μm. Producing these casts is a multi-step process: chamber design, interior structure print and cure (resin print), exterior structure (mould box) print (filament print), pre-casting preparation, PDMS pour and polymerization, mould and coverslip cleaning, and plasma fusion. A separate protocol for each step is included below.

Chamber Design

Chambers need to be designed in 3D modelling programs, and designed at-scale. While many programs are available, TinkerCad is free and easy to use, while other programs are more capable. Chambers should be sized to the width of the coverslip (perhaps 1-2 mm smaller), and should cover most or all of the length of the coverslip. Keep in mind that PDMS is flexible, but can tear, so "walls" between larger features need to be at least 1 mm thick (preferably thicker) to avoid tearing while removing from the mould. If a secure seal is required between two areas, a coverslip contact area of at least 2 mm is required. The resin-printed part of the mould should contain the finer features, but should be printed separate from the "walls" of the casting chamber, as for all but the smallest of features, removable mould walls are needed to be able to extract the cast chamber.

For the walls, design a FDM-printed box which will seal tightly to the resin mould - typically, the "hole" in the wall should be ~0.5 mm wider/longer than the resin-printed insert. The height of the box will determine the thickness of the cast - thicknesses of 2.5 to 5 mm are a good balance between having a thick enough chamber versus having a chamber which can be easily extracted from the mould.

DLP (resin) Printing

Export the resin component of the mould as a steriolithography (STL) file and import/slice it in Chitobox or Lychee slicer. Try to limit the use of supports that contact the part of the mould which will contact the PDMS, as these can leave blemishes on the cast chamber. Print, ideally using a white or semi-transparent material, remove any supports, and cure thoroughly post-print. If you do not know how to print using the resin printer, please get training prior to attempting it on your own.

Following printing, the residual photocatalysts must be driven from the mould. A failure to perform this step will prevent the PDMS from polymerizing. This is achieved by placing the printed mould on a hot plate set to 65C, or in an oven at the same temperature, and heating for at least 18 hours. If using a hot plate, and if its possible without damaging the mould, flip the mould half way through the curing process to heat from both sides of the mould. Do not heat above 65C as this can warp the mould. At this time we have tested conventional, ABS-like and water-washable resins using this approach, and it has worked for all three resin types.

FDM (filament) Printing

The wall portion of the mould needs to be printed in a material resistant to temperatures over 60C, and which has some flexibility. PETG is the best material for this use that we have identified to-date. The print resolution of this component of the mould is not critical, but we recommend 0.2 mm layer heights with a 0.4 mm nozzel to ensure an accurate print. Aside from a isopropyl alcohol and water rinse post-print to remove any adhesive, no special treatments are required to prepare this mould component for casting.

Pre-Casting Preparation

1. Assemble the mould by press-fitting the resin print into the FDM walls.
   1. If the fit is too tight, carefully sand the sides of the resin print.
   2. If the fit is too loose, re-print the walls using a slightly smaller opening for the resin print.
   3. Small gaps can be filled with VALAP (1:1:1 mix of vaseline, lanolin and paraffin), but take care to not get VALAP on the mould surface (e.g. seal gaps from the bottom of the mould, not within the mould cavity itself).
2. Once assembled, spray a light coating of mould release onto the mould.
   1. Rotate the mould during the process to ensure all surfaces receive a light coating.

The mould is now ready for casting.

PDMS Pour

1. Measure out a 10:1 ratio of PDMS and catalyst, preparing 10-20% more than required.
   1. Prepare in a 15 ml or 50 ml centrifuge tube.
   2. To limit waste, cast multiple moulds at once.
2. Mix the PDMS/catalyst thoroughly - once mixed, bubbles should appear throughout the PDMS. Bubble-free areas are likely unmixed, and may leave unpolymerized areas in your cast.
3. Degas the PDMS by centrifugation for 2 minutes at the maximum safe speed of the centrifuge (~4,000 RPM for the benchtop centrifuge in the Heit lab).
4. Place the moulds into a petri dish or other container to limit spills, and carefully fill with the degassed PDMS. Avoid creating bubbles during the pour.
5. Place the moulds into a vacuum chamber and bring to a vacuum use the wall-vacuum source. Let sit 1-2 minutes, then repressurize to burst any bubbles.
6. Reapply the vacuum for 20 minutes.
7. Transfer the chambers, in the spill-containment container, to a 37C incubator and incubate overnight. Alternatively, let polymnerize for 2 days at room temperature. This will generate a soft cure.
8. Separate the walls from the underlying resin mould, then separate the chamber from the resin mould.
9. Using scissors, trim off any overflow from the cast.

Mould and Coverslip Cleaning

Clean the chamber:

1. Rinse in isopropyl alcohol, scrubbing with gloved hands to remove any VALAP, PDMS particles and other material.
2. Rinse with dH2O from the white tap.
3. Spray lightly with ethanol and dry using a lint-free kimwipe.
4. Store between kimwipes until assembly.

It is recommended that you assemble the chamber immediately after cleaning the cast, but casts can be stored for days prior to assembly.

Clean the coverslips:

1. Clean, follow our standard Acid Washing Coverslips protocol. These can be stored indefinitely before use
2. Before use, rinse off any dust with distilled water and dry using a kimwipe.

Plasma Fusion

Gloves need to be worn throughout this procedure as residual oils on the chamber or coverslip will impair fusion.

Coverslip Preparation (perform first)

1. Place the coverslip(s) into the plasma cleaning chamber.
2. Evacuate the chamber by running the vacuum pump for ~10 seconds.
3. Fill the chamber with oxygen.
4. Evacuate the chamber by running the vacuum pump for ~10 seconds.
5. Fill the chamber with oxygen.
6. Evacuate the chamber by running the vacuum pump for ~20 seconds.
7. Close the valve on the chamber and transfer it to the microwave.
8. Using the microwave at maximum power, expose the coverslip to 2 seconds of oxygen plasma.
   1. Oxygen plasma will produce a white/grey glow
   2. Air/nitrogen plasma is pink/purple; if you see this oxygen purging of the chamber did not work. Dispose of the coverslip.
9. Remove the coverslip and place on a kimwipe or other dust-free surface, being certain to keep the plasma-exposed face upwards.

Chamber Preparation (perform second)

1. Place a PDMS chamber into the plasma cleaning chamber, placing the coverslip-facing face upwards so that it is exposed to the plasma.
2. Perform steps 2-8 from the coverslip preparation procedure
   1. If you do not get the characteristic colour of oxygen plasma, the cast is ruined and will not fuse to the coverslip.
3. Remove the cast from the chamber and place on a kimwipe with the coverslip-contacting surface facing upwards.
4. Without touching the coverslip to the cast, carefully align the coverslip, being sure that the plasma-treated surfaces are facing each other.
   1. Once aligned, "roll" the coverslip onto the cast such that no air is trapped between the cast and the coverslip (see note 1).
   2. Press firmly to ensure good contact is made (see note 2).
5. Transfer the assembly to a hot plate at 120 C, coverslip against the hot plate, and let cure for 45-60 min. If possible, a small weight can be placed on top of the chamber to help ensure even and complete fusion.

Note 1: If alignment is difficult a small drop of methanol can be placed on the cast; this will allow the coverslip to slide on the cast without adhering.

Note 2:Dry contact between the coverslip and chamber will lead to an immediate covalent fusion between chamber and coverslip. Do not try to move/;tear them apart after contact as this will permanently damage the adhesion and prevent proper chamber assembly.

Post-Processing

While the chambers prepared above are ready for immediate use, there are some post-processing steps that can be performed:

• Chambers can be autoclaved to sterilize them. Place then in an autoclave-safe container, between kimwipes or gauze, and autoclave using the instrument setting.
• Chambers can be exposed to ~5 seconds of air plasma to alter the surface charge of the chamber and coverslip. This may make coating with some reagents work better, may improve compatibility with lipids, and may improve the adhesion of some cell types.
• Luer connectors, liquid lines, etc, can be added. These components are best attached using either the inherent stickiness of the PDMS, or using a fresh mixture of PDMS + curing agent as a glue. If using PDMS as a glue, the glue can be cured in 2 hours at 65C on a hot plate.
• Cleaning can be performed with almost any solvent desired. Recommended cleaning agents include 0.1% triton x-100 in PBS or TBS, or 70% ethanol.
• The glass coverslip can be coated with proteins or other substances using your normal protocols, and profusion the solution into the chamber area(s) to be coated.