The increasing demand for high-resolution, high-throughput biological imaging has led to complex and expensive microscopes. Long-term observation of living organisms with minimal behavioral impact requires controlled environments, often necessitating incubator integration. The complexity of modern microscopes necessitates specialized expertise for assembly, maintenance, and data analysis, creating a barrier for many researchers. While tailored commercial solutions exist, they are often costly, inflexible, and poorly documented, limiting their adaptability to various tasks. This lack of accessibility hinders reproducibility and contributes to the scientific reproducibility crisis. The complexity of optical setups, with components from diverse manufacturers adhering to varying standards, further complicates customization. Therefore, an open standard for microscope components is crucial to facilitate versatile, easily adaptable imaging instruments accessible to a wider range of users. Existing open-source projects like Flamingo, lattice light sheet, and open-SPIM have made progress in this area, along with other low-cost solutions such as the open-flexure stage and Foldscope. The widespread accessibility of 3D printing and consumer electronics now allows for the creation of such an open standard, where software and algorithmic solutions can compensate for limitations in inexpensive components. The UC2 (You. See. Too.) approach aims to establish this open standard, relying on a modular, 3D-printable framework, open-source software, and comprehensive documentation to provide cost-effective and versatile microscopic imaging.

The paper reviews existing advanced microscopy techniques and the challenges associated with their cost, complexity, and accessibility. It highlights the growing reproducibility crisis in science and proposes open-source solutions as a remedy. Several open-source microscopy projects are cited, including Flamingo, lattice light sheet microscopy, open-SPIM, open-flexure stage, and Foldscope. The authors also mention previous attempts at creating modular opto-mechanical toolboxes, such as uCube, and highlight the potential of 3D printing and consumer electronics for building low-cost, high-performance microscopy systems. Existing image processing methods such as deconvolution and quantitative phase imaging are discussed as means to compensate for limitations of low-cost components.

The UC2 toolbox utilizes a 4f optical system design, implemented with a 3D-printable framework of modular cubes (50mm design pitch). These cubes are designed for easy assembly using a magnetic snap-fit mechanism, allowing flexible arrangement of components. The system is designed to interface with off-the-shelf components and existing equipment from companies such as Thorlabs. A module developer kit (MDK) facilitates the creation of custom inserts. The system can scale from simple setups to complex systems like light-sheet microscopes. Consumer electronics such as cameras, motors, and microcontrollers (Arduino and ESP32) are integrated to allow smart microscopy capabilities and remote control via wired or wireless communication (I2C, WiFi, MQTT). Power is supplied through conductive magnets or wires. The paper details the creation of an incubator-enclosed bright-field microscope, utilizing a low-cost objective lens, a Raspberry Pi camera, and an LED array for illumination. This microscope was used for a 168-hour imaging session of monocyte-to-macrophage differentiation (four systems, parallel experiment). The same basic setup was adapted into a light-sheet microscope by adding a laser pointer, a second objective lens, a beam expander, a cylindrical lens, and a larger baseplate. Other applications demonstrated include wide-field fluorescence microscopy, image scanning microscopy (ISM), intensity diffraction tomography, and structured illumination. A cellphone camera was also integrated in place of the Raspberry Pi camera for improved sensitivity. Image processing was performed using custom Python scripts, Fiji, and ImJoy. Sample preparation involved isolating peripheral blood mononuclear cells (PBMCs) and culturing them to observe differentiation. Image analysis involved drift correction, background correction, flat fielding, and cell size measurements in Fiji. Quantitative phase imaging used aIDT methods. Statistical analysis used one-way ANOVA with Tukey's correction.

The UC2 toolbox successfully produced a functional incubator-enclosed bright-field microscope capable of long-term (7 days) imaging of cellular processes like macrophage differentiation. The study demonstrates the reproducible results of the method using four identical systems. The microscope's compact size and parallelization capabilities allow high-throughput experiments at a low cost. The same core system was effectively adapted into a light-sheet fluorescence microscope, demonstrating its versatility, although this setup's resolution was limited by the camera used. Using a cellphone camera instead of the Raspberry Pi camera significantly improved image resolution in fluorescence imaging. The practical resolution using a cellphone camera was determined to be 0.6 µm, superior to the resolution obtained with the Raspberry Pi camera (1.13 µm). The UC2 system was shown to be capable of various imaging modalities including wide-field fluorescence, structured illumination, image scanning microscopy, intensity diffraction tomography, and quantitative phase imaging (aIDT and qDPC), showcasing its versatility. The findings on macrophage differentiation replicated previous work, revealing the elongated morphology of macrophages correlated with their movement.

The UC2 toolbox addresses the need for an accessible and versatile open-source microscopy platform. Its modularity and 3D-printable design make it highly customizable and adaptable to various imaging techniques, significantly lowering the cost of entry into advanced microscopy. The successful implementation of both brightfield and light-sheet microscopy within the UC2 framework highlights the practicality of this open standard. The results demonstrate the potential for reproducible, low-cost research by making complex microscopy techniques easily available to both researchers and educators. The use of readily available components and open-source software promotes broader accessibility and collaboration within the scientific community, directly addressing the reproducibility crisis. The limitations of the 3D-printed components (thermal stability) were addressed through iterative design and algorithmic solutions like autofocus.

The UC2 toolbox presents a significant advancement in open-source microscopy, providing a low-cost, highly versatile, and easily customizable platform. Its modular design and open-source nature address the critical needs for accessibility and reproducibility in microscopy research and education. Future development could focus on improving the thermal stability of 3D-printed components, exploring novel imaging modalities, and expanding the range of compatible components. The integration of more advanced sensors and computational methods could further enhance the system's capabilities and broaden its applications.

The long-term stability of 3D-printed parts made from PLA and ABS can be affected by temperature fluctuations. Although addressed through design iterations and software-based autofocus, this remains a potential limitation. The resolution of the light-sheet microscope was limited by the sensitivity of the Raspberry Pi camera, making it more suitable for educational purposes than high-resolution research. The use of low-cost components may introduce some limitations in performance compared to high-end commercial systems.