vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.Super-resolution microscopy based on three-dimensional single-molecule localization microscopy (3D-SMLM) is now well established 1,2 , and its widespread adoption has led to the development of more than 36 software packages dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching 3 . While the initial emphasis in the 3D-SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, high-fidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (visualization of the local universe in a micro environment, pronounced 'volume'), an immersive virtual reality (VR)-based visualization software package purposefully designed to render large 3D-SMLM datasets. It is free for academic use. vLUME enables robust visualization, segmentation, annotation and quantification of millions of fluorescence puncta from any 3D-SMLM technique. vLUME has an intuitive user interface and is compatible with all commercial gaming VR hardware (Oculus Rift/Rift S and HTC Vive/Vive Pro; Supplementary Video 1). Although other microscopy data (that is, confocal) visualization tools have previously explored VR technology using volumetric representations 4,5 , vLUME has been specifically and purposefully created for SMLM. It accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics. (A comparison with other VR and non-VR tools can be found in Supplementary Table 1.)vLUME is a point-cloud based 3D-SMLM data visualization tool able to render all pointillism-based multidimensional datasets. It differs from other 3D tools for 3D-SMLM visualization such as ViSP 6 by providing a complete VR interactive environment and intuitive interface for life scientists, dedicated to data visualization, segmentation and analysis. Users load multidimensional particle-list datasets into vLUME (.csv files; Fig. 1a), such as those generated by commonly used 3D-SMLM software 7,8 . This allows users to comprehend the spatial and temporal relation between

Super-Resolution (SR) Microscopy based on 3D Single-Molecule Localization Microscopy (SMLM) is now well established 1,2 and its wide-spread adoption has led to the development of more than 36 software packages, dedicated to quantitative evaluation of the spatial and temporal detection of fluorophore photoswitching 3 . While the initial emphasis in the 3D SMLM field has clearly been on improving resolution and data quality, there is now a marked absence of 3D visualization approaches that enable the straightforward, highfidelity exploration of this type of data. Inspired by the horological phosphorescence points that illuminate watch-faces in the dark, we present vLUME (Visualization of the Universe in a Micro Environment, pronounced 'volume') a free-for-academic-use immersive virtual reality-based (VR) visualization software package purposefully designed to render large 3D-SMLM data sets. vLUME enables robust visualization, segmentation and quantification of millions of fluorescence puncta from any 3D SMLM technique. vLUME has an intuitive user-interface and is compatible with all commercial VR hardware (Oculus Rift/Quest and HTC Vive, Supplementary Video 1). vLUME accelerates the analysis of highly complex 3D point-cloud data and the rapid identification of defects that are otherwise neglected in global quality metrics.