![]() Figure 1d shows the resulting SMLM reconstruction. Next two-dimensional (2D) or three-dimensional (3D) spatial images, captured using the astigmatism method ( Huang et al., 2008 Zhang et al., 2015), can be processed using ThunderSTORM. RainbowSTORM first requires the images to be cropped for spatial and spectral analysis. The general workflow for RainbowSTORM analysis is outlined in Figure 1c. In addition to providing a flexible calibration tool, RainbowSTORM also includes a sSMLM analysis module for processing sSMLM images ( Fig. 1b). Scale bars: 1 μm 2.2 sSMLM image processing Reconstructions of three separate channels showing ( f) mitochondria labeled with AF647, ( g) microtubules labeled with CF660, ( h) peroxisomes labeled with CF680 and ( i) the overlay of the three channels. ( c) RainbowSTORM workflow showing how the system calibration module interacts with the analysis module, ( d) SMLM reconstruction and ( e) Pseudocolored sSMLM reconstruction. ( b) sSMLM images with the spatial and spectral images captured on different parts of a detector. ( a) Conceptual schematic of a general sSMLM system. calibration lamps or multiple laser lines) and multicolor fluorescent beads. Calibration in RainbowSTORM can be performed using both calibrated light sources (e.g. ![]() While grating-based systems are calibrated by linearly fitting pixel positions to known wavelengths ( Dong et al., 2016), prism-based systems are calibrated using second-order ( Huang et al., 2018) or third-order ( Zhang et al., 2015) polynomial fittings. RainbowSTORM calibrates sSMLM images acquired using different systems, where the dispersive element ( Fig. 1a) can be either a grating or a prism. ![]() 2 Features and methods 2.1 System calibration Derivations for spectroscopic analysis ( Song et al., 2018) and flowcharts of the algorithms used in RainbowSTORM are included in the Supplementary Information (SI). We provide a detailed user guide that includes descriptions and workflows for the processes implemented in RainbowSTORM. Multicolor images can be generated by setting different user-defined spectral centroid ranges for channels with predefined colors. RainbowSTORM uses the spectral centroids (or intensity-weighted spectral means) of each localized stochastic event to define a range of spectral colors and render pseudocolored super-resolution images ( Bongiovanni et al., 2016 Dong et al., 2016 Zhang et al., 2015). RainbowSTORM leverages the functionality of the existing SMLM processing tool ThunderSTORM ( Ovesny et al., 2014) to attain spatial information while providing crucial spectroscopic tools for system calibration as well as spectral identification and classification. Here, we present RainbowSTORM, an open-source spectroscopic analysis plug-in for ImageJ/FIJI. While a variety of software algorithms and packages are currently available for processing and analyzing traditional SMLM images ( Sage et al., 2019), software tools for comprehensive spectroscopic analysis of sSMLM images remain unavailable. Overall, sSMLM shows great promise to further extend existing SMLM. For example, sSMLM detected the polarity of the environment surrounding dye molecules ( Bongiovanni et al., 2016) and enabled the discovery of previously undetected molecular conformations of dyes ( Kim et al., 2017). sSMLM has also led to new functional imaging capabilities through the analysis of variations in the spectra of individual molecules. Thus far, sSMLM has enabled multicolor imaging ( Zhang et al., 2015) and tracking ( Huang et al., 2018) of as many as four different fluorescent species using a single excitation source. Recently, spectroscopic SMLM (sSMLM) ( Bongiovanni et al., 2016 Dong et al., 2016 Zhang et al., 2015), which simultaneously detects the location and full emission spectra of each emission event was reported. Single-molecule localization microscopy (SMLM) ( Betzig et al., 2006 Rust et al., 2006 Sharonov and Hochstrasser, 2006) overcomes the optical diffraction limit by localizing stochastically emitting fluorescent molecules with high localization precision (typically 10–20 nm).
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