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Related Experiment Video

Updated: Apr 12, 2026

High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

Published on: October 28, 2025

Post-processing strategies in image scanning microscopy.

J E McGregor¹, C A Mitchell¹, N A Hartell¹

¹Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 7RH, UK.

Methods (San Diego, Calif.)

|May 13, 2015

Summary

This summary is machine-generated.

Image scanning microscopy (ISM) achieves improved resolution by scanning point-wise and reassigning pixels. This study optimizes computational processing for better signal recovery, especially when imaging through scattering media like tissue.

Keywords:

Image formation Optical microscopy Pinhole Pixel reassignment Point spread function Super-resolution

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Area of Science:

Microscopy
Optical Imaging
Biophysics

Background:

Image scanning microscopy (ISM) enhances resolution by a factor of √2 over conventional widefield microscopy.
Pixel reassignment, achieved optically or computationally, doubles the highest accessible spatial frequency.
Pinhole use, physical or digital, is common in ISM to reject out-of-focus light.

Purpose of the Study:

To simulate and analyze Image Scanning Microscopy (ISM) datasets using various processing techniques.
To address challenges in computational pixel reassignment and pinholing for improved image quality.
To investigate the impact of tissue scattering on excitation foci and develop robust signal recovery methods.

Main Methods:

Simulation of an ISM dataset using a test image for computational analysis.
Application of standard and non-standard processing methods, including pixel reassignment and digital pinholing.
Utilizing localization software to identify fluorescence maxima for accurate pinhole centering and signal recovery.

Main Results:

Demonstrated achievement of the predicted resolution improvement with standard pixel reassignment on simulated data.
Quantified the effects of realistic displacements between reference and true excitation positions.
Developed and validated a strategy using detected fluorescence maxima for accurate pinhole alignment, recovering degraded signal.

Conclusions:

Optimized computational processing, particularly accurate pinhole alignment to detected maxima, significantly improves ISM image quality.
The developed strategy effectively recovers signal compromised by inaccurate reference grids or displacements, even in challenging conditions.
Findings are validated with experimental data from multiphoton ISM, showing robust performance in recovering signal across the field of view, even after tissue penetration.