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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Monolithic dual-wedge prism-based spectroscopic single-molecule localization microscopy.

Ki-Hee Song1, Benjamin Brenner1, Wei-Hong Yeo1

  • 1Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Rd., Evanston, IL, 60208, USA.

Nanophotonics (Berlin, Germany)
|July 25, 2022
PubMed
Summary
This summary is machine-generated.

A novel dual-wedge prism spectrometer enhances spectroscopic single-molecule localization microscopy (sSMLM). This innovation improves spatial and spectral precision for advanced cellular imaging applications.

Keywords:
Single-Molecule Localization MicroscopySpectroscopySuper-resolution Fluorescence Imaging

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Area of Science:

  • Optical microscopy
  • Spectroscopy
  • Nanotechnology

Background:

  • Spectroscopic single-molecule localization microscopy (sSMLM) enables simultaneous spectroscopic analysis and imaging of single molecules.
  • Traditional sSMLM methods using discrete optical components face challenges like non-uniform spectral dispersion, high transmission loss, and complex alignment.
  • These limitations hinder the precision and performance of sSMLM techniques.

Purpose of the Study:

  • To overcome the limitations of conventional sSMLM by developing a new imaging spectrometer.
  • To enhance the spatial and spectral precision of sSMLM for improved molecular analysis.
  • To demonstrate a compact and robust sSMLM system with reduced alignment demands.

Main Methods:

  • Designed and optimized a dual-wedge prism (DWP)-based monolithic imaging spectrometer.
  • Utilized ray-tracing and numerical simulations to verify DWP performance.
  • Experimentally validated the DWP-sSMLM system by imaging nanospheres and cellular structures.

Main Results:

  • The DWP design achieved spectrally dispersed focused beams with minimal deviation and wavefront error.
  • The monolithic assembly minimized transmission loss and optical alignment complexity.
  • DWP-sSMLM demonstrated significantly improved spatial and spectral precision compared to grating-based sSMLM.

Conclusions:

  • The DWP-based imaging spectrometer effectively addresses key challenges in sSMLM.
  • DWP-sSMLM offers superior precision for high-throughput single-molecular analysis.
  • The developed system is suitable for advanced applications like 3D multicolor cellular imaging.