Spectroscopic single-molecule localization microscopy (sSMLM) combines super-resolution microscopy and spectroscopy. Its single molecule sensitivity and high spectral precision have made it uniquely valuable for several applications, including multicolor imaging, chemical characterization, polarity sensing, and multiplexed single particle tracking. However, widespread adoption is hampered by a lack of standardization in optical implementation, calibration techniques, and image processing. We demonstrate our lab’s efforts to develop tools that simplify adoption and optimize photon efficiency, including protocols for calibration techniques, a user-friendly imageJ plugin for image processing, and a fabricated monolithic beam splitter and prism designed to fit into a microscope body with minimal optical alignment.
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