Related Experiment Video
Updated: Sep 5, 2025
09:45
Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
25.4K
Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative
Nazar Oleksiievets1, Christeena Mathew2, Jan Christoph Thiele1
1III. Institute of Physics - Biophysics, Georg August University, 37077 Göttingen, Germany.
Nano Letters
|July 6, 2022
Summary
This study compares wide-field and confocal fluorescence-lifetime single-molecule localization microscopy (FL-SMLM) techniques. Both methods offer high resolution and lifetime information for advanced bioimaging, with practical guidance provided for experimental design.
Area of Science:
- Super-resolution microscopy
- Biophotonics
- Advanced imaging techniques
Background:
- Fluorescence-lifetime single-molecule localization microscopy (FL-SMLM) integrates high resolution with fluorescence lifetime information.
- FL-SMLM can be implemented using either confocal-laser scanning microscopy (CLSM) or wide-field microscopy.
- A systematic comparison of these two FL-SMLM approaches is needed to guide experimental design.
Purpose of the Study:
- To systematically compare confocal-laser scanning microscopy (CLSM)-based and wide-field FL-SMLM.
- To evaluate the performance of both FL-SMLM approaches in different spectral regions.
- To provide practical advice for designing FL-SMLM experiments.
Main Methods:
- Comparison of a commercial wide-field FL-SMLM system with a home-built CLSM-based FL-SMLM system.
- Utilized metal-induced energy transfer (MIET) for 3D single-emitter localization.
- Employed DNA-PAINT for lifetime-based multiplexed bioimaging.
Main Results:
- Characterized the performance of both wide-field and confocal FL-SMLM systems.
- Demonstrated 3D localization capabilities using FL-SMLM combined with MIET.
- Showcased multiplexed bioimaging using DNA-PAINT with FL-SMLM.
Conclusions:
- Both wide-field and confocal FL-SMLM are viable techniques for super-resolution imaging with lifetime information.
- The choice between wide-field and confocal FL-SMLM depends on specific experimental requirements.
- This study offers practical insights for optimizing FL-SMLM experimental design.
Related Concept Videos
01:16Confocal Fluorescence Microscopy
13.9K
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
13.9K
01:37Super-resolution Fluorescence Microscopy
7.4K
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
7.4K
01:28Three-Dimensional Microscopy in Microbiology
276
Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...
276
01:29Two-Dimensional Microscopy in Microbiology
461
Two-dimensional (2D) microscopy encompasses a range of optical techniques that capture images within a single focal plane, offering detailed representations of microscopic structures. These techniques are essential in biological and medical research, enabling the visualization of cellular and subcellular structures with different levels of contrast and specificity.There are several major types of 2D microscopy, each with strengths and applications.Bright-Field MicroscopyBright-field microscopy...
461