Journal ArticleTWINKLE: An open-source two-photon microscope for teaching and researchSchottdorf, Manuel (ORCID:0000000254684255); Rich, P Dylan; Diamanti, E Mika; Lin, Albert (ORCID:0000000245415889); Tafazoli, Sina; Nieh, Edward H (ORCID:0000000321546224); Thiberge, Stephan Y (ORCID:0000000265836613)Pesce, Luca<p>Many laboratories use two-photon microscopy through commercial suppliers, or homemade designs of considerable complexity. The integrated nature of these systems complicates customization, troubleshooting, and training on the principles of two-photon microscopy. Here, we present “Twinkle”: a microscope for Two-photon Imaging in Neuroscience, and Kit for Learning and Education. It is a fully open, high performing and easy-to-set-up microscope that can effectively be used for both education and research. The instrument features a >1 mm field of view, using a modern objective with 3 mm working distance and 2 inch diameter optics combined with GaAsP photomultiplier tubes to maximize the fluorescence signal. We document our experiences using this system as a teaching tool in several two week long workshops, exemplify scientific use cases, and conclude with a broader note on the place of our work in the growing space of open scientific instrumentation.</p>plos.org2025-02-1310672190PLOS ONE202e03189241932-6203https://doi.org/10.1371/journal.pone.03189241734030National Science Foundation