Work

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

Year: 2026

Type: article

Abstract: 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

Source: Communications Biology

DOI: 10.1038/s42003-026-10035-z

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

Institutions: Harvard University, Massachusetts General Hospital

Language: English

Topic: Advanced Fluorescence Microscopy Techniques

Subfield: Biophysics

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $4,190