Design, assembly, alignment and application of a versatile, open-source, single-pixel microscope

Single-pixel microscopy (SPM) is an emerging imaging technique in which a sample is illuminated with a series of micro-structured light patterns, typically generated by a digital micromirror device (DMD). After interaction with the sample, light is collected by a bucket detector, and the image is reconstructed through computational algorithms, such as basis transformations or compressive sensing. DMD achievable framerates and wide spectral range has allowed SPM to develop a wide range of applications, including polarization state analysis, phase imaging, and fluorescence lifetime measurements. To achieve optimal performance in these applications, a precise system configuration is required ensuring the effective projection of the structured light patterns. Nevertheless, the incorporation of a DMD introduces additional complexity, particularly in alignment, which can significantly affect system performance if not properly addressed. This work presents a comprehensive framework for the design, assembly, and alignment of a modular, open-source SPM system. The proposed procedures minimize aberrations introduced during construction and ensures the accurate projection of structured light patterns onto the sample. The modular design facilitates integration across multiple illumination sources and enables simultaneous brightfield transmission and reflection imaging. The proposed system achieves resolution near the diffraction limit, surpassing previous SPM configurations without requiring numerical or optical enhancement techniques. Performance validation through imaging experiments on both biological and non-biological samples demonstrates the system’s robustness and versatility. By providing detailed design and assembly instructions, this work contributes to the openness and reproducibility of SPM and serves as a valuable resource for researchers aiming to build high-performance, customizable single-pixel imaging systems.