Related Experiment Video

Updated: Feb 5, 2026

Fluorescence Lifetime Macro Imager for Biomedical Applications
06:01

Fluorescence Lifetime Macro Imager for Biomedical Applications

Published on: April 7, 2023

1.2K

Bright GFP with subnanosecond fluorescence lifetime

Anastasia V Mamontova1, Ilya D Solovyev2,3, Alexander P Savitsky2,3

  • 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Scientific Reports
|September 7, 2018
PubMed

Insights

Researchers developed BrUSLEE, a novel fluorescent protein probe with a subnanosecond fluorescence lifetime and high brightness. This breakthrough enhances multiparameter imaging using fluorescence lifetime imaging microscopy (FLIM) by enabling better discrimination between probes.

Area of Science:

  • Biochemistry
  • Microscopy
  • Molecular Biology

Background:

  • Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for multiparameter imaging.
  • Current GFP-like fluorescent proteins have limited fluorescence lifetimes (2.3-3.5 ns), restricting their use in advanced FLIM applications.
  • A broader range of fluorescence lifetimes is needed for improved probe differentiation in FLIM.

Purpose of the Study:

  • To engineer a novel fluorescent protein (FP) probe with a subnanosecond fluorescence lifetime and high brightness.
  • To overcome the limitations of existing FPs for multiparameter FLIM.
  • To enable clearer signal distinction for spectrally similar probes.

Main Methods:

  • Semi-rational amino acid substitution selection was employed.
  • Key positions (Thr65, Tyr145, Phe165) were mutated based on their known effects on fluorescence lifetime (FL) and excited state electron transfer.
  • The engineered triple mutant was named BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter).

Main Results:

  • A new FP probe, BrUSLEE, was created with a subnanosecond fluorescence lifetime.
  • BrUSLEE exhibits exceptional fluorescence brightness, reaching 80% of EGFP.
  • The probe demonstrated high performance in multiparameter FLIM experiments, providing distinguishable signals from common GFPs.

Conclusions:

  • The developed BrUSLEE probe significantly expands the utility of multiparameter FLIM.
  • Amino acid substitutions at critical positions can alter the equilibrium of radiative and non-radiative excited state processes.
  • BrUSLEE offers a valuable tool for advanced biological imaging requiring precise probe differentiation.

More Related Videos

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells09:45

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Published on: February 9, 2012

25.9K
Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy09:30

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Published on: January 18, 2017

12.5K

Related Concept Videos

Photoluminescence: Fluorescence and Phosphorescence01:23

Photoluminescence: Fluorescence and Phosphorescence

Photoluminescence is a process where a molecule absorbs light energy and re-emits it in the form of light. This phenomenon occurs when a substance absorbs photons, promoting its electrons to higher energy level excited states, followed by a relaxation process in which the electrons return to their original ground state energy levels and emit light. Photoluminescence is widely observed in various materials, including semiconductors, and organic and inorganic compounds.
A pair of electrons in a...
3.9K
Atomic Fluorescence Spectroscopy01:29

Atomic Fluorescence Spectroscopy

Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which...
976
Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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,...
21.1K
Variables Affecting Phosphorescence and Fluorescence01:26

Variables Affecting Phosphorescence and Fluorescence

Fluorescence and phosphorescence are essential phenomena in fields like analytical chemistry, biological imaging, and materials science, where they detect molecular properties and visualize cellular structures. Understanding the variables that influence these luminescent behaviors is crucial for maximizing accuracy and efficiency in their applications. These variables can broadly be grouped into chemical structure, solvent properties, and external conditions, each playing a distinct role in...
1.5K
Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.
1.6K
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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...
14.6K