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Updated: Jan 11, 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, CNY149, 13th St, Charlestown, 100190, MA, USA.

Research Square

|November 19, 2025

Summary

This summary is machine-generated.

We developed a new differential confocal microscopy method using pinhole engineering to improve imaging resolution and depth. This technique offers simpler, high-resolution fluorescence lifetime imaging microscopy (FLIM) for biological studies.

Keywords:

Confocal FLIM STED Super-resolution

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Last Updated: Jan 11, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

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 research.
Fluorescence Lifetime Imaging Microscopy (FLIM) provides quantitative molecular information but often requires complex setups.

Purpose of the Study:

To enhance lateral resolution and axial sectioning depth in differential confocal microscopy.
To develop a simpler, super-resolution Fluorescence Lifetime Imaging Microscopy (FLIM) technique.
To enable high-resolution, multiplexed FLIM for advanced subcellular imaging.

Main Methods:

Implemented pinhole engineering in differential confocal microscopy.
Integrated an intensity-weighted lifetime imaging strategy.
Validated the technique through simulations and experiments on 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 leading super-resolution FLIM techniques with simpler implementation.

Conclusions:

The novel differential confocal microscopy technique significantly improves imaging performance.
The integrated intensity-weighted lifetime imaging strategy offers an accessible route to super-resolution FLIM.
This method advances high-resolution, multiplexed imaging for biological applications.