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SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole.

Elisabetta Di Franco1, Angelita Costantino2,3,4, Elena Cerutti1,5

  • 1Department of Physics and Astronomy "Ettore Majorana", University of Catania, Via S. Sofia, 64, 95123, Catania, Italy.

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Summary

A new software, SPLIT-pinhole (SPLIT-PIN), enhances optical sectioning in microscopy by analyzing images with tunable pinhole sizes. This method reduces out-of-focus background without sacrificing signal quality, improving image clarity.

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Area of Science:

  • Microscopy
  • Biophotonics
  • Image Processing

Background:

  • Point-scanning microscopy uses a detection pinhole for optical sectioning, rejecting out-of-focus light.
  • Minimizing pinhole size theoretically maximizes optical sectioning but reduces signal, creating a trade-off between resolution and signal-to-noise ratio.

Purpose of the Study:

  • To introduce and describe SPLIT-pinhole (SPLIT-PIN) software for enhanced optical sectioning.
  • To provide a method for reducing out-of-focus background in microscopy images without compromising signal quality.

Main Methods:

  • The SPLIT-PIN method applies the separation of photons by lifetime tuning (SPLIT) algorithm to image series acquired with a tunable pinhole.
  • Software processes at least two images acquired using commercially available microscopes with tunable pinholes (confocal, STED).

Main Results:

  • SPLIT-PIN software generates images with significantly reduced out-of-focus background.
  • Demonstrated applicability across various imaging modalities, including confocal, super-resolution STED, and live organoid imaging.

Conclusions:

  • SPLIT-PIN offers an effective alternative to traditional pinhole-based optical sectioning.
  • The software enables improved image quality and axial resolution in diverse microscopy applications.