3D Printed Microscopy Chambers

Customized imaging chambers can be 3D printed using FDM or SLA printing technology, and attached to a #1.5 thickness coverglass to allow for high-resolution imaging. When prepared properly, these chambers show no cytotoxicity or other signs of altered cell behaviour, even in cells cultured for multiple days. These chamber designs used in the Heit Lab, and are the newest version of the chambers presented in our publication in Biochemistry and Cell Biology, modified based on some exciting advances in the Mod3D preprint by the Truant Lab.

In addition to directly printing chambers, 3D printing can also be used to make moulds for casting PDMS chambers, as described in our publication in Biochemistry and Cell Biology.

Chamber Designs

Chambers are available to fit 18 mm x 18 mm and 24 mm x 50 mm coverslips, with different well arrangements. We are still testing some of these designs, but the files can be downloaded at the following link. The images below show the file name, chamber layout, culture areas, and recommended media volumes for the varying chambers.

• 18 mm chambers
• 24 x 50 mm chambers

Baseplates

These baseplates are designed to hold the 18 mm or 50 mm chambers in a Chamlide TC Live Cell Chamber. These should be printed in black PETG for temperature resistance and durability, 0.2 mm layer height. The bottom plates require supports, and it helps to iron (smooth) all top surfaces. The small circular inserts hold 4 mm diameter, 1 mm thick, disk magnets. These hold the top-plates in-place during imaging. To ensure the lid and base magnets lineup properly, a magnet alignment tool is provided. Glue 4 mm diameter x 1 mm thick disk magnets onto either end of the alignment tool, ensuring that the pole of the magnet on the 'B' end is the opposite pole of the magnet on the 'L' end (e.g. North for 'B', South for 'L'). Use the tool to hold onto, and place, magnets into the lid ('L') and Base/Bottom ('B'). Place a small drop of instant (cyanoacrylate) glue into each circular insert, and then use the 'L' magnet to hold and place the magnets for the lids, and the 'B' magnet to hold and place the magnets for the base. This will ensure that the magnets in the base will attract the magnets in the lid, holding the chamber together. The STL files for both stages and the magnet alignment tool can be downloaded here.

Printing Chambers - FDM (Filament) Printing

FDM printing is the easiest in terms of printing and post-printing processing, and it is easier to find biocomatable materials. It is important to use a good quality filament free of potentially cytotoxic or fluorescent compounds - we recommend a black or unpigmented, FDA-food grade certified PLA or PETG filament such as those sold by Filaments.ca.

When printing, be certain that the coverslip-contacting part of the print faces upwards.

Recommended Print Settings

• 0.4 mm diameter nozzle
• 100% infill
• 0.1 mm layer height
• Print speed/acceleration set for high quality prints (varies with printer)
• Ironing:
  • Top-most surface for chambers (this helps with the bond to the coverslip)
  • None on lids or alignment guides
• No supports

FDM Printing

1. Layout your desired prints using PrusaSlicer (or your preferred slicing software). Be certain to position the prints such that the surface that will contact the coverslip faces upwards. The numbers and letters on the chamber will be upside-down if the chamber is positioned correctly.
2. Configure the print settings as above and slice.
3. Print
4. Carefully remove the print from the print bed and wash for 15 min in distilled water.
5. Dry chamber before assembly (see below).

Printing Chambers - DLP (Resin) Printing

Only resin currently validated for cell-contact use.

DLP (resin) printing provides superior resolution and a flatter surface for bonding the coverslip to the chamber. However, the prints must be treated carefully after printing to remove any residual polymer or photocatalyst, as these are highly cytotoxic. At this time the only resin we've found that works for cell-contact purposes eSun's eResin-PLA Bio-Photopolymer Resin (Black).

Recommended Print Settings

• Maximum x/y resolution
• No anti-aliasing
• 0.05 mm layer height
• Exposure times need to be optimized for individual instruments, but for our Elegoo Mars 3:
  • 4 burn-in layers @ 60 s
    • 0 transition layers
    • 4 s light-off delay
    • 6 mm lift
    • 0.4 mm/m lift speed
    • 150 mm/m retract speed
  • Normal layers:
    • 0.05 mm layer height
    • 8.5 s exposure
    • 0 mm lift distance
    • 65 mm/m lift speed
    • 150 mm/m retract speed
    • 0 light-off delay

Correct orientation, rotation, & supports

DLP Printing

1. Layout prints as desired in Lychee Slicer or Chitubox (or your preferred slicing software). Be certain to position the prints such that the surface that will contact the coverslip face upwards. The numbers and letters on the chamber will be upside-down if the chamber is positioned correctly.
2. Rotate the prints 40° along the long axis (see image to right).
3. Use autosupports to lift and generate supports for the chambers. A 3 to 5 mm lift is recommended.
4. Manually remove any supports that contact the surface that will contact the coverslip as this surface needs to be absolutely smooth.
5. Print
6. The prints are soft at this point, so treat them gently for the next few steps.
7. Carefully remove the print from the print bed and place in a wash container of isopropyl alcohol. It is fine to use isopropyl that has been used previously for mashing prints in this step.
8. Using flat cutters carefully remove all supports.
9. Place the chambers in a ultrasonic cleaner. Do not stack the prints. Cover completely with clean (previously unused) isopropyl alcohol. Clean for 90 seconds.
10. Carefully remove the chambers and rinse with cold tap water. Then place the prints in the curing station, coverslip-contact side up. Position the chambers such that they do not contact each other. Cure for 20 minutes, then flip the parts and cure an additional 20 minutes. Do not crowd parts during this step - it is better to cure in multiple batches than it is to have shadowed regions that do no cure.

Chamber Assembly

Assembly is the same whether the chambers are printed via DLP or FDM.

1. Acid wash coverslips in advance of assembly day, ensuring they are completely dry and dust free before assembly.
2. Place a coverslip into the bottom of the alignment guide.
3. Apply the glue:
   1. Option A: Paint a thin layer of SS-433T silicone glue (Silicone Solutions, OH, USA) onto the coverslip-contacting surface of the chamber. This provides a stronger bond, but may be less biocompatable than PDMS.
   2. Option B: Mix and degass PDMS, using a 10:1 polymer:catalyst ratio. Paint a thin layer onto the coverslip-contacting surface of the chamber.
4. Put the chamber (glue-side down) into the alignment guide and press onto the coverslip for 10 seconds.
5. Carefully invert the alignment guide and remove the chamber.
6. Check the seal - a good seal will appear wet; any dry-appearing areas are not glued. If you see a dry area, gently press that part of the chamber with your finger. If a good seal cannot be made, remove the coverslip and repeat steps 2-6 with a new coverslip. This step is critical, as poorly glued chambers will leak.
7. Transfer the completed chambers to a humidified, 37°C incubator for 24 hours to cure.

Optional: The strength of the PDMS bond can be increased by treating the chambers with an ~3 second exposure to oxygen plasma after step 7.

Chamber Cleaning & Sanitation

These chambers cannot be fully sterilized, but can be sanitized. As such, all cultures should be performed using media containing anitbiotic/anitmycotic solution.

1. Soak the chambers, with gentle agitation, ddH₂O, for 30 minutes. Replace the water and soak with agitation for an additional 30 minutes.
2. Dry using a slow flow of air from a compressed air valve.
3. Place into small zip-seal bags, with a matching lid, and seal.
4. Place in the UV crosslinker, being certain to not overlap/shadow any parts. Irradiate for 5,000 mJ, then flip the chambers and irradiate for another 5,000 mJ.

3D Printing Moulds for Casting PDMS Chambers

Both FDM and DLP can be used to prepare PDMS moulds, which can be used to cast PDMS chambers. FDM requires the minimal preparation, but has lower resolution than DLP.

FDM Printed PDMS Moulds

1. Print moulds in any material, using a minimal layer height.
2. Wash for 15 minutes in water, then dry.
3. The prints are now ready for casting.

DLP Printed PDMS Moulds

1. Print moulds using any water-washable resin at maximum resolution.
2. Carefully remove the print from the print bed and place in a wash container full of distilled water.
3. Drain the wash container, then remove the supports from the part.
4. Place the parts back in a wash container, and cover completely with clean (previously unused) distilled water. Wash with gently shaking for 20 minutes.
5. Carefully remove the moulds and place in the curing station. Position the moulds such that they do not contact each other. Cure for 20 minutes, then flip the parts and cure an additional 20 minutes. Do not crowd parts during this step - it is better to cure in multiple batches than it is to have shadowed regions that do no cure.
6. Place the moulds in a vacuum chamber and draw a full vacuum (<1 torr). Place vacuum chamber in a 37°C incubator for 24 hours. This will eliminate any volatile that can prevent the PDMS from curing.

Casting the Chambers

1. In a centrifuge tube prepare ~20% more PDMS than you expect to need, mixing a 1:10 ratio of curing agent:polymer. Mix thoroughly.
2. Centrifuge at maximum speed (4,000 RPM) for 5 minutes to degas the PDMS.
3. While the PDMS is degasing, assemble the moulds and coat in a thin layer of food-safe release agent.
4. Pour the PDMS into the mould. A light vacuum can be used to help elminate any bubbles that form during casting. Do not use a hard vacuum as this will draw gas from inside the mould, which will then create inclusions in the PDMS.
5. Place in a 37°C incubator for 24 hours to cure.
6. Remove the chamber from the mould, trim off any flashing, and then wash in 70% ethanol with gently agitation, for 15 minutes.

Assembling PDMS Chambers

1. Acid wash coverslips in advance of assembly day, ensuring they are completely dry and dust free before assembly.
2. Place the chamber and coverslip into a glass vacuum chamber, being certain to keep them from touching.
3. Draw a hard vacuum (<1 torr, 10-15 second run with the pump) and refill the vacuum chamber with oxygen.
4. Repeat step 3 two additional times to ensure the chamber is fully purged of air and contains only oxygen.
5. Draw a hard vacuum (<0.1 torr) by running the pump for ~30 seconds, then close off vacuum chamber.
6. Place vacuum chamber into the microwave and microwave on high for 3 seconds after the plasma ignites. Note: plasma should be pink-grey in colour. If violet in colour there may be too much nitrogen and a poor seal may result.
7. Carefully remove the coverslip from the vacuum chamber and place on a clean kimwipe.
8. Carefully remove the PDMS chamber from the vacuum chamber, and in a single smooth motion, push it against the coverslip. Do this starting from one side of the chamber and then "rolling" it onto the coverslip - this avoids trapping air within the seal.
9. Press firmly for 20 seconds, then place on a hot-plate at 120°C for an hour. This should covalently bond the coverslip to the PDMS.
10. These chambers can be autoclaved to steralize.