Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

12.3K
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...
12.3K
Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

1.9K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
1.9K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Side-by-Side Systematic Characterization of Novel FPs in Budding Yeast.

ACS synthetic biology·2026
Same author

An mTurquoise2-Based Glucose Biosensor.

ACS sensors·2026
Same author

A green lifetime biosensor for calcium that remains bright over its full dynamic range.

eLife·2025
Same author

Leukocytes use endothelial membrane tunnels to extravasate the vasculature.

Cell reports·2025
Same author

Fluorescence Fluctuation Analysis of Arabidopsis thaliana Somatic Embryogenesis Receptor-Like Kinase and Brassinosteroid Insensitive 1 Receptor Oligomerization.

Biophysical journal·2024
Same author

On the glow of cremated remains: long-lived green photo-luminescence of heat-treated human bones.

Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology·2024
Same journal

Mapping the 3D Chromosome Organization of a Biosynthetic Gene Cluster by Capture Hi-C (CHi-C).

Methods in molecular biology (Clifton, N.J.)·2026
Same journal

Mapping the 3D Chromosome Organization of Streptomyces by Hi-C.

Methods in molecular biology (Clifton, N.J.)·2026
Same journal

CUT&Tag Epigenomic Profiling of Biosynthetic Gene Clusters in Arabidopsis thaliana.

Methods in molecular biology (Clifton, N.J.)·2026
Same journal

Rhizobium rhizogenes-Mediated Hairy Root Transformation Protocol for Lotus japonicus and Other Legumes.

Methods in molecular biology (Clifton, N.J.)·2026
Same journal

Characterization of Bioactive Saponins from Sea Cucumbers.

Methods in molecular biology (Clifton, N.J.)·2026
Same journal

Methods for Functional Validation of Terpenoid Metabolic Clusters in Nicotiana benthamiana and Aspergillus oryzae.

Methods in molecular biology (Clifton, N.J.)·2026
See all related articles

Related Experiment Video

Updated: May 7, 2026

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
11:51

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

14.0K

Optimization of fluorescent proteins.

Daphne S Bindels1, Joachim Goedhart, Mark A Hink

  • 1Swammerdam Institute for Life Sciences, van Leeuwenhoek Centre for Advanced Microscopy, Section of Molecular Cytology, University of Amsterdam, Amsterdam, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)
|October 11, 2013
PubMed
Summary
This summary is machine-generated.

Researchers are developing new fluorescent proteins (FPs) with improved brightness and photostability for advanced microscopy. This optimization enhances biomolecular studies using fluorescence spectroscopy and imaging techniques.

More Related Videos

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast
09:23

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

Published on: November 28, 2018

6.5K
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
14:26

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

Published on: January 19, 2011

12.5K

Related Experiment Videos

Last Updated: May 7, 2026

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
11:51

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Published on: April 27, 2018

14.0K
Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast
09:23

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

Published on: November 28, 2018

6.5K
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
14:26

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

Published on: January 19, 2011

12.5K

Area of Science:

  • Biophysics
  • Molecular Biology
  • Microscopy

Background:

  • Fluorescent proteins (FPs) are crucial tools for studying biomolecules in situ, enabling high-resolution imaging.
  • Existing FP variants offer diverse colors and photoswitchable properties, expanding fluorescence spectroscopy and microscopy capabilities.
  • Further improvements in FP brightness, photostability, and compatibility with biophysical techniques are needed for advanced applications.

Purpose of the Study:

  • To describe key characteristics of fluorescent proteins (FPs) that determine their application.
  • To present experimental protocols for optimizing FP properties.
  • To detail a three-step optimization procedure for developing novel FP variants.

Main Methods:

  • FP optimization involves random or site-directed mutagenesis of template sequences.
  • Primary screening of FP libraries in bacteria assesses fluorescence intensity, lifetime, and emission spectra.
  • Promising mutants undergo in vitro characterization of key properties and in vivo evaluation in mammalian cells.

Main Results:

  • The study outlines a systematic approach to engineer and characterize novel fluorescent protein variants.
  • Optimization protocols focus on enhancing brightness, photostability, and spectral properties.
  • In vivo assessments in mammalian cells evaluate FP performance in a biological context.

Conclusions:

  • Optimized fluorescent proteins are essential for advancing fluorescence spectroscopy and microscopy techniques.
  • The described multi-step optimization procedure allows for the development of FPs with tailored characteristics.
  • Improved FPs will enhance the study of biomolecules with greater resolution, specificity, and sensitivity.