Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.
Koen J A MartensMartijn GobesEmmanouil ArchontakisRoger R BrillasNiels ZijlstraLorenzo AlbertazziJohannes HohlbeinPublished in: Nano letters (2022)
Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1 st diffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0 th and 1 st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.
Keyphrases
- single molecule
- living cells
- optical coherence tomography
- low cost
- light emitting
- atomic force microscopy
- high resolution
- deep learning
- machine learning
- primary care
- healthcare
- health information
- dual energy
- photodynamic therapy
- high speed
- magnetic resonance imaging
- quality improvement
- social media
- heavy metals
- real time pcr