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Structured detection for simultaneous super-resolution and optical sectioning in laser scanning microscopy.

Alessandro Zunino1, Giacomo Garrè1,2, Eleonora Perego1,3

  • 1Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy.

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|August 18, 2025
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Summary
This summary is machine-generated.

This study introduces a new image scanning microscopy method that reconstructs super-resolution images from single-plane acquisitions. It enhances optical sectioning and signal-to-noise ratio, overcoming limitations of current techniques for thick samples.

Keywords:
Fluorescence imagingImaging and sensingSuper-resolution microscopy

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Area of Science:

  • Optical Microscopy
  • Biophotonics
  • Image Processing

Background:

  • Image scanning microscopy (ISM) offers improved resolution and signal-to-noise ratio (SNR) over confocal microscopy.
  • Current ISM methods struggle with thick samples, lacking optical sectioning and introducing a trade-off between sectioning and SNR.
  • Limitations arise from detector size constraints in achieving optical sectioning in thick samples.

Purpose of the Study:

  • To develop a novel method for image scanning microscopy that overcomes existing limitations in optical sectioning and SNR.
  • To achieve digital and optical super-resolution, high SNR, and enhanced optical sectioning from single-plane acquisitions.
  • To generalize the reconstruction algorithm for fluorescence lifetime imaging (FLI).

Main Methods:

  • A straightforward reconstruction algorithm was designed to invert the physical model of ISM image formation.
  • The method leverages the inherent axial information embedded in detector array acquisitions.
  • Validation was performed using a custom setup with a single-photon avalanche diode array detector on biological samples.

Main Results:

  • The proposed method reconstructs images with digital and optical super-resolution, high SNR, and enhanced optical sectioning.
  • Feasibility demonstrated in both linear and nonlinear fluorescence excitation regimes.
  • The algorithm was generalized for FLI, utilizing the timing capabilities of the detector array.

Conclusions:

  • The developed method effectively overcomes the limitations of conventional ISM, particularly for thick samples.
  • It provides superior performance compared to existing reconstruction techniques.
  • The approach is extensible to other laser scanning microscopy techniques and advanced imaging modalities like FLI.