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Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
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Fluorescence emission difference microscopy by superoscillation excitation.

Yong Liu1,2, Xiaona Wang2, Shaocong Liu2

  • 1College of Electronics and Information Engineering, Shanghai University of Electric Power, Shanghai, China.

Journal of Microscopy
|March 26, 2019
PubMed
Summary

Superoscillation excitation enhances fluorescence emission difference (FED) microscopy resolution. This superresolution technique improves imaging by using specialized light spots, achieving up to 2x better resolution than confocal microscopy.

Keywords:
Fluorescence emission difference microscopysuperoscillationsuperresolution

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Area of Science:

  • Optical Microscopy
  • Superresolution Imaging
  • Diffraction Theory

Background:

  • Fluorescence emission difference (FED) microscopy offers high-resolution imaging by subtracting images from two confocal modes.
  • Current FED microscopy typically uses standard excitation spots, limiting potential resolution enhancements.
  • Superoscillation excitation, capable of creating sub-wavelength focal spots, has not been applied to FED microscopy.

Purpose of the Study:

  • To investigate the application of superoscillation excitation in FED microscopy for enhanced spatial resolution.
  • To explore the generation of solid and donut superoscillation excitation spots using radially polarized Laguerre-Gaussian beams.

Main Methods:

  • Utilized radially polarized Laguerre-Gaussian (LG) beams with spiral phase modulation to generate solid and donut superoscillation excitation spots.
  • Employed vector diffraction theory and simulations to analyze the performance of the proposed FED microscopy.
  • Investigated the effectiveness of the two-view Richardson-Lucy (RL) deconvolution method in mitigating side lobe effects.

Main Results:

  • Superoscillation excitation significantly enhances the spatial resolution of FED microscopy.
  • Achieved approximately 2x resolution improvement compared to general confocal microscopy with optimized LG3,1 beam parameters (pupil radius ratio 0.85, subtractive factor 0.3).
  • Demonstrated a 1.1x resolution enhancement compared to general FED microscopy.
  • The two-view RL deconvolution method effectively reduced side lobe impacts related to the subtractive factor and pinhole size.

Conclusions:

  • Superoscillation excitation is a viable method for improving FED microscopy resolution.
  • The proposed FED microscopy with superoscillation excitation surpasses the resolution of conventional confocal and FED microscopy.
  • Two-view RL deconvolution is crucial for managing artifacts and maximizing the benefits of superoscillation excitation in FED microscopy.