Spectral Phasor Applied to Spectrally-Resolved Single Molecule Localization Microscopy

Spectrally-resolved single-molecule localization microscopy (srSMLM) has emerged as a powerful tool for exploring the spectral properties of single emitters in localization microscopy. By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super-resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two-dimensional space, preserving key spectral characteristics for single-molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual-color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram-positive Enterococcus hirae in response to gramicidin exposure, a membrane-perturbing antibiotic treatment. Spectral phasor provides a robust, model-free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi-color spectrally-resolved single-molecule imaging.