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<p>In this article, we present a differential confocal microscopy technique based on pinhole engineering that significantly enhances both lateral resolution and axial sectioning depth. Through simulations and experimental validation with a fluorescent calibration slide, we demonstrated a 1.6-fold improvement in lateral resolution and a two-fold increase in axial sectioning capability. Building on these advancements, we integrated an intensity-weighted lifetime imaging strategy to surpass the diffraction limit in fluorescence lifetime measurements. This approach achieved high spatial resolution and quantitative lifetime data comparable to leading super-resolution FLIM (Fluorescence Lifetime Imaging Microscopy) techniques, yet allows for simpler implementation. We further validated the method in subcellular structure lifetime imaging, demonstrating improved resolution and axial depth enhanced lifetime-based multiplexing capacity. This new method provides an accessible route to high-resolution, multiplexed FLIM for advanced biological imaging.</p>

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Pinhole engineering based enhanced resolution (PEER) for fluorescence lifetime imaging microscopy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月14日

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  • Wonsang Hwang,
  • Sinyoung Jeong,
  • J. Matthew Dubach,
  • Conor L. Evans,
  • Iván Coto Hernández

摘要

In this article, we present a differential confocal microscopy technique based on pinhole engineering that significantly enhances both lateral resolution and axial sectioning depth. Through simulations and experimental validation with a fluorescent calibration slide, we demonstrated a 1.6-fold improvement in lateral resolution and a two-fold increase in axial sectioning capability. Building on these advancements, we integrated an intensity-weighted lifetime imaging strategy to surpass the diffraction limit in fluorescence lifetime measurements. This approach achieved high spatial resolution and quantitative lifetime data comparable to leading super-resolution FLIM (Fluorescence Lifetime Imaging Microscopy) techniques, yet allows for simpler implementation. We further validated the method in subcellular structure lifetime imaging, demonstrating improved resolution and axial depth enhanced lifetime-based multiplexing capacity. This new method provides an accessible route to high-resolution, multiplexed FLIM for advanced biological imaging.