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Related Experiment Video

Updated: Apr 15, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

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

Wonsang Hwang¹, Sinyoung Jeong², J Matthew Dubach³

¹Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA.

Communications Biology

|April 13, 2026

Summary

This summary is machine-generated.

We developed a new differential confocal microscopy method using pinhole engineering. This technique improves imaging resolution and depth, offering an accessible way for high-resolution, multiplexed fluorescence lifetime imaging microscopy (FLIM) in biology.

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Last Updated: Apr 15, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Published on: February 9, 2012

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Published on: October 28, 2018

Fluorescence Lifetime Macro Imager for Biomedical Applications

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Area of Science:

Microscopy
Biophotonics
Super-resolution imaging

Background:

Confocal microscopy offers optical sectioning but is limited by diffraction.
Improving lateral resolution and axial depth in microscopy is crucial for biological studies.
Fluorescence Lifetime Imaging Microscopy (FLIM) provides molecular information but often requires complex super-resolution techniques.

Purpose of the Study:

To enhance lateral resolution and axial sectioning depth in confocal microscopy.
To develop a simpler, high-resolution alternative to existing super-resolution FLIM techniques.
To enable advanced biological imaging with improved resolution and multiplexing capabilities.

Main Methods:

Differential confocal microscopy with pinhole engineering.
Intensity-weighted lifetime imaging strategy.
Simulations and experimental validation using fluorescent calibration slides and subcellular structures.

Main Results:

Achieved a 1.6-fold improvement in lateral resolution.
Demonstrated a two-fold increase in axial sectioning capability.
Obtained quantitative lifetime data comparable to super-resolution FLIM with simpler implementation.

Conclusions:

The developed technique offers enhanced resolution and axial depth in confocal microscopy.
This method provides an accessible route to high-resolution, multiplexed FLIM for biological imaging.
The approach surpasses the diffraction limit for fluorescence lifetime measurements.