Related Experiment Video
Updated: Jun 2, 2026
11:03
How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells
Published on: January 7, 2019
Two-photon absorption properties of fluorescent proteins
Mikhail Drobizhev1, Nikolay S Makarov, Shane E Tillo
1Department of Physics, Montana State University, Bozeman, Montana, USA. drobizhev@physics.montana.edu
Nature Methods
|April 30, 2011
Summary
Researchers need brighter fluorescent proteins for two-photon imaging. This review guides selecting the best fluorescent proteins and excitation wavelengths for optimal imaging of living systems.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Two-photon excitation microscopy offers advantages for deep tissue imaging and reduced phototoxicity in live biological samples.
- Selecting appropriate fluorescent proteins (FPs) is crucial for maximizing signal and minimizing photobleaching in two-photon applications.
- Existing guides often lack comprehensive data on newer far-red FPs, limiting choices for advanced imaging.
Purpose of the Study:
- To provide a comprehensive review of two-photon absorption properties for a wide range of fluorescent proteins.
- To guide researchers in selecting the brightest FPs and optimal excitation wavelengths for their specific two-photon imaging needs.
- To include data on novel far-red fluorescent proteins for extended spectral applications.
Main Methods:
- Literature review and compilation of published two-photon absorption cross-section data for various FPs.
- Analysis of spectral properties, including excitation and emission maxima, and quantum yields.
- Categorization of FPs based on their performance in two-photon excitation.
Main Results:
- Identification of several highly efficient FPs with large two-photon cross-sections across different spectral ranges.
- Detailed comparison of brightness and photostability for commonly used and novel FPs.
- Recommendations for excitation wavelengths that maximize signal for specific FPs, including far-red variants.
Conclusions:
- The choice of FP significantly impacts the success of two-photon imaging in living systems.
- This review serves as a practical resource for optimizing FP selection and excitation parameters.
- Future development should focus on FPs with enhanced brightness and photostability in the far-red spectrum.
More Related Videos
Related Concept Videos
01:19Protein Dynamics in Living Cells
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...
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...
01:26Variables 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...
01:23Photoluminescence: 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...
A pair of electrons in a...
01:25Fluorescence 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.
01:14Photoluminescence: Applications
Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...
01:29Two-Dimensional Microscopy in Microbiology
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...