bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.121608; this version posted May 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. The Incubot: A 3D Printer-Based Microscope for Long-Term Live Cell Imaging within a Tissue Culture Incubator George O. T. Mercesa, b, 1, Conor Kennedya, b, Blanca Lenocia, b, Emmanuel G. Reynaudc, Niamh Burkea, b, and Mark Pickeringa, b aSchool of Medicine, University College Dublin, Co. Dublin, Republic of Ireland, D04 V1W8. bUCD Centre for Biomedical Engineering, University College Dublin, Co. Dublin, Republic of Ireland, D04 V1W8 cSchool off Biomolecular and biomedical Science, University College Dublin, Co. Dublin, Republic of Ireland, D04 V1W8 1Corresponding Author Abstract to deal with the significant cost is to arrange a consortium or multi-group effort to purchase the equipment, sharing Commercial live cell imaging systems represent a large the resource after purchase, or institutional investment in financial burden to research groups, while current open core imaging facilities. In the case of shared equipment, source incubator microscopy systems lack adaptability and long-term studies can prove difficult to schedule and can are sometimes inadequate for complex imaging experimen- reduce flexibility in experimental design, particularly in tation. We present here a low-cost microscope designed early stage, exploratory and scoping studies. Usage fees for inclusion within a conventional tissue culture incubator. applied to maintain the equipment may also then limit access The build is constructed using an entry level 3D printer as to lower income laboratories. This cost barrier to owning the basis for the motion control system, with Raspberry Pi complex imaging equipment can stifle research efforts in the imaging and software integration, allowing for reflected, biomedical field. oblique, and fluorescence imaging of live cell monolayers. The open source movement within science has been pro- The open source nature of the design is aimed to facilitate viding innovations in affordable laboratory equipment, adaptation by both the community at large and by individual including microscopy and optics (6–9). Open source ap- researchers/groups. The development of an adaptable and proaches for low-cost incubator based microscopes have easy-to-use graphic user interface (GUI) allows for the been explored (10–12) and serve as great examples of scientist to be at the core of experimental design. Simple affordable microscopes for live-cell imaging. However, modifications of the base GUI code, or generation of an several limitations with current designs curtail their potential entirely purpose-built script, will allow microscopists to widespread use within laboratories. The use of a CNC ma- place their experimental design as the priority, as opposed chine is neither common nor accessible amongst biomedical to designing experiments to fit their current equipment. The researchers. Options that allow for in-incubator microscopy build can be constructed for a cost of roughly C1000 and but lack a motion control system result in a low-throughput. thus serves as a low-cost and adaptable addition to the open The use of only transmitted white light reduces the range source microscopy community. of potential applications of such builds. An ideal scenario Microscopy | Tissue Culture | Low-Cost | Python | Raspberry Pi | Live-Cell would be an affordable open source system that makes use of Imaging conventional low-cost commercial products while allowing Correspondence: [email protected] for adaptability as a group’s research interests or capabilities may change. Hardware in Context Microscopy is always a trade-off between field of view (FOV) and resolution. A static microscope is simple and Live imaging of cells under physiological conditions is an effective, but only allows a small FOV to be imaged. essential technique in biomedical sciences for analyses of Imaging more than a single FOV requires some element cell proliferation (1,2), cell migration (3,4), and cell-cell of motion control of wither the optics or the sample. The interactions (5). Two commercial options are available open flexure microscope is a remarkable solution to this to researchers aiming to perform live-cell imaging. First, problem, as it includes very precise motion control at a low purchase a specific stage incubator designed to upgrade cost (13). The main drawback is that the range of movement the current expensive microscopy system you already own. is limited and do not allow for scanning plates or flasks Alternatively, purchase a whole new system designed only that are routinely used in cell culture experiments. Luckily, to be use within a tissue culture incubator, often with a the problem of precise and repeatable control of three- significant price tag, with additional costs for any add-ons dimensional movement has been solved in another field: 3D such as “automated scanning”. Despite the widespread use printing. Commercial 3D printers require sub-millimeter of live imaging, the cost of the equipment necessary for precision over a range of travel of tens of centimeters. such types of experiments remain high. A common way Merces et al. | bioRχiv | May 29, 2020 | 1–23 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.121608; this version posted May 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Graphical Abstract Therefore, we designed an incubator microscope, the In- physiological conditions and avoids the negative impact of cubot, which repurposes the core motion control system thermal drift associated with heated stages. from a simple, entry-level cartesian 3D printer (a Tronxy A Graphical User Interface (GUI) was developed using X1). An associated easy-read assembly guide has been made Kivy for Python 3, allowing for simple user control over available on the online repository to help explain all steps parameters for live cell imaging, including the number of in construction of this system, with the aim that non-experts time points to image over, the temporal spacing between in microscopy or optics can construct and use this equipment. them, the number and layout of wells, the area of each well imaged, and automated focusing for each well. The total cost of this system (C1061.79) represents a substantial saving over commercial equipment (14). Hardware Description This build represents a low-cost alternative to commercial The Incubot uses a Tronxy X1 3D printer as a motorised microscopy systems for physiological imaging of cells, with stage for optics movement allowing for XYZ imaging of specific utility in simple imaging experimentations. The use tissue culture flasks or plates (Fig 1.). The X and Z axes are of simple components and 3D printing for construction of coupled directly to the Y axis to allow for the 3-dimensional the build allow for modification and adaptation of the build movement of the optics unit which is coupled to the extruder for individual users based on their needs. plate. A tissue culture plate is maintained above the optics via a stage composed of 15x15 mm aluminium extrusion and Potential uses of the Incubot: 3D-printed components, which remains stationary while the optics are translated. Moving optics under a static sample • Long-term monitoring of tissue culture experiments prevents movement of the medium during imaging, which • Long-term monitoring of tissue culture flasks may affect the cells. The optics unit is composed of 1” ø for determination of optimum time for passag- tubes, connecting a commercial infinite-conjugate objective ing/experimentation lens to a Raspberry Pi Camera (V2), using an achromatic doublet tube lens to allow for microscopic image acquisition. • Semi-portable device for tissue culture information The optional inclusion of a long pass filter allows for fluores- outreach cent imaging of samples. A silicone moulded rig was used to mount 8 LEDs around the objective lens, and thus allow • Use as a regular scanning microscope for fixed cell for several illumination techniques to be performed, such imaging as reflected, oblique, and fluorescent. Both the motorised stage and the plate-holder are mounted onto a 300 mm x 300 Design Files Summary mm breadboard (M6) with vibration-absorbing feet. The whole unit can be placed within a tissue culture incubator All files relating to the Incubot can be found at our public for long term use. This setup allows for inverted imaging of online repository: https://osf.io/es3hr/ a tissue culture plate/flask within an incubator maintained at 2 | bioRχiv Merces et al. | The Incubot bioRxiv preprint doi: https://doi.org/10.1101/2020.05.28.121608; this version posted May 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Figure 1. A) Fully Assembled Incubot within a Tissue Culture Incubator with Key Components/Features Annotated. B) Representative images of HeLa-GFP live cells within an incubator using the Incubot using oblique white illumination (Oblique), blue LED excitation of GFP (Fluorescence) and the two images overlain (Overlay). Scale bar indicates 500 µm. XYZ_Coupler Plate_Holder 3D printed unit designed to couple the X and Z axes of the 3D printed unit to allow for stable elevation of a tissue original Tronxy X1 to its Y axis. culture plate over the optics of the Incubot for imaging. Optics_Holder MotionValidationProtocol 3D printed unit to replace the extruder portion of the original Python script to perform sequential positive and negative Tronxy X1.