STORM imaging buffer with refractive index matched to standard immersion oil

Abstract Stochastic optical reconstruction microscopy (STORM) provides exceptional super-resolution imaging by sparsely blinking individual dye molecules in thiol-containing media. STORM is now well-established for imaging thin biological specimens, and recent technological advancements have expanded its use to thick tissues. While the use of mounting media with an oil refractive index has been shown to reduce light scattering within tissues and thus greatly improve imaging depth and resolution in optical microscopy, the refractive index of STORM imaging buffers is typically water-like and oil-index (OI) buffers have never been considered for this purpose. In this study, we report a 3-pyridinemethanol-based STORM buffer that matches the refractive index of standard immersion oil. Our OI buffer exhibits similar superior performance in terms of photoswitching of Alexa Flour 647 dye and STORM image quality in fixed cells as conventional STORM buffers, despite having a completely different refractive index. Interestingly, it shows remarkable stability for at least 25 days, and potentially longer, which will enable STORM imaging of a large number of cells on a single prepared slide, as well as larger field-of-view imaging through multiple field stitching. By achieving perfect index matching with oil immersion objectives, OI buffers can produce accurate nanoscale morphology of thin biological specimens, without the need for complex microscope calibrations across sample depth. More importantly, our STORM buffer is expected to play a crucial role in lightsheet STORM applications for thick tissues by reducing light scattering, thereby leading to improved imaging depth and localization performance.