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Updated: May 21, 2025

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

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Exploiting the detector distance information in image scanning microscopy by phasor-based SPLIT-ISM.

Elisabetta Di Franco^1,2,3, Giulia Tedeschi³, Lorenzo Scipioni³

¹Department of Physics and Astronomy "Ettore Majorana", University of Catania, Catania, Italy.

Biomedical Optics Express

|March 20, 2025

Summary

This summary is machine-generated.

Image scanning microscopy (ISM) resolution is enhanced by combining it with separation of photons by lifetime tuning (SPLIT). This novel SPLIT-ISM technique improves bio-imaging resolution and optical sectioning on existing hardware.

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Area of Science:

Biophotonics
Microscopy
Super-resolution imaging

Background:

Confocal microscopy uses a pinhole to improve resolution but is limited by signal loss.
Image scanning microscopy (ISM) overcomes this with detector arrays and pixel reassignment.
Separation of photons by lifetime tuning (SPLIT) is a super-resolution technique using fluorescence lifetime analysis.

Purpose of the Study:

To enhance the resolution of image scanning microscopy (ISM) by integrating it with the separation of photons by lifetime tuning (SPLIT) technique.
To investigate the impact of SPLIT-ISM on lateral resolution and optical sectioning.
To validate SPLIT-ISM performance on commercially available ISM systems.

Main Methods:

Developed SPLIT-ISM by analyzing information from array detector distances within ISM.
Applied SPLIT-ISM to biological images acquired using Genoa Instruments PRISM and Zeiss Airyscan ISM systems.
Evaluated SPLIT-ISM performance using the QuICS algorithm to quantify resolution and SNR.

Main Results:

SPLIT-ISM achieved an additional 1.3x increase in lateral resolution compared to standard pixel-reassigned ISM.
The technique demonstrated a concomitant increase in optical sectioning capabilities.
Quantitative analysis confirmed improved spatial resolution and SNR for both tested ISM systems.

Conclusions:

SPLIT-ISM effectively enhances the resolution and optical sectioning of existing ISM systems.
The integration of SPLIT with ISM offers a significant advancement in bio-imaging capabilities.
SPLIT-ISM provides a valuable tool for high-resolution biological imaging applications.