Our lab uses a flow cell developed by Thomas Hauling and Hamish Forrest. We use it for in situ sequencing but it could also be useful for other forms of spatial profiling.
In the lab, we perform In Situ Sequencing (ISS), a technique that relies on hundreds of barcodes, i.e. DNA sequences that are read in a microscope using dye-conjugated probes. To increase the number of distinguishable barcodes, we employ multiple rounds of imaging. In each round, the dyes of the preceding imaging round are removed (stripping) and probes for the new round are added (labeling).
To ease this process we developed a flow cell that allows stripping and labelling of the specimen while it remains mounted under the microscope. This flow cell could be useful not only for ISS but also for spatially resolving in situ profiling of analytes (e.g. nucleic acids, proteins). Below we share its design and give instructions for how to build it.
The CAD design for the Flow Cell is available as an STL file.
The Flow Cell is designed to be used with 2 mm (1.6 mm lumen) tubing and a peristaltic pump. Assembly requires Norland Optical Adhesive (UV cement), type 81, and a source of UV light. UV cement was chosen for its transparency, ease of application (it does not expand on curing), strength following curing and lack of reactivity to tested and planned staining techniques.Tube placement and base cover slip
1. Check your 3D print for imperfections which may affect channel width and flatness of model. Clean the work surface (with RNAase Zap for in-situ sequencing).
2. Clip 2mm diameter tubing into the channels provided. Tubing protruding into the inner chamber is necessary to avoid UV cement blocking the lumen later on, try to limit protrusion to a few millimetres.
3. Flip the model and apply UV cement directly onto the tubing through the circular holes. These function to ensure that tubing is completely surrounded by UV cement. Add more UV cement around the border of the chamber leaving no gaps.
4. Place the base 22 x 50 mm cover slide onto the model using the guiding walls.
5. Use UV light to cure the cement. Prolonged exposure can crack the cover slip, so allow dwell time between bursts. Check if UV cement is completely dry by pressing the slip into the model. Add extra UV cement to edges of the cover slip to ensure a seal, and cure.
Tissue-mounted cover slip
6. Flip the model and apply a small amount of UV cement to the wall of the inner oval chamber and exposed tubing, so as to form a complete cement ring. Ensure that UV cement does not enter the lumen of the tubing.
7. Place the cover slip (tissue side) down on the cement. Perfect alignment is not essential, but the entire ring-shaped ridge must contact the slide to provide a seal, indicated by UV cement against glass. Without a complete sealing ring of UV cement, cement can enter the chamber later and flow into tubing. When curing with UV light, cover the sample with tin foil if it is photosensitive.
8. Add UV cement into the wells indicated by white arrows. The UV cement will spread underneath the overhanging cover slip and eventually fill the moat. Once the wells are close to overflowing, the moat is filled.
9. Cure the UV cement, allowing time for glass to cool between bursts. Curing the thick layer of UV cement can take several long bursts, use pressure to indicate that the cement is completely cured. An extra layer of cement on the cover slip border and over the exposed tubing will strengthen the seal.
We welcome your feedback on how to improve these instructions. Please send comments to Hamish Forrest or Thomas Hauling.