Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires provision of appropriate experimental conditions in a format that meets the microscopy’s optical requirements. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized live-cell imaging chambers that are compatible with a range of imaging modalities including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and which supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting PDMS microfluidic devices. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with modern microscopy techniques.

While new high-resolution microscopy techniques are continually developed, adoption of these methods is often difficult due to an inability to meet the experimental conditions required for an experiment in a format which also meets the demanding optical requirements of these microscopy techniques. Although specialized imaging chambers can meet these challenges, the difficulty of manufacturing customized chambers in-house and the relatively high cost and design inflexibility of commercial chambers has limited the incorporation of imaging chambers into fluorescence and super-resolution microscopy experiments. Herein, we demonstrate the use of fused deposition modeling (3D printing) for producing inexpensive, customized imaging chambers that are compatible with long-duration live-cell imaging using fluorescence and super-resolution microscopy techniques. In this approach, biocompatible 3D printing plastics are used to generate imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip suitable for fluorescence and super-resolution imaging. This technique produces a chamber that is impermeant to liquids that can support the growth and imaging of cells over multiple days. The utility of these chambers is then demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach to produce imaging chambers that are compatible with many modern microscopy techniques.