Flashbody: A Next Generation Fluobody with Fluorescence Intensity Enhanced by Antigen Binding
Devina Wongso1, Jinhua Dong2, Hiroshi Ueda2
1Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS) , 11 Biopolis Way #05-02 Helios, Singapore 138667, Singapore.
Analytical Chemistry
|May 24, 2017
Summary
Researchers developed a novel "Flashbody" probe for visualizing molecules in cells. This genetically encoded antibody probe exhibits antigen-dependent fluorescence, enhancing imaging capabilities for cellular dynamics.
Area of Science:
- Biotechnology
- Molecular Biology
- Cell Biology
Background:
- Fluorescent probes are essential for observing molecular dynamics in living cells.
- Genetically encoded probes offer advantages for in vivo imaging.
- Existing probes may have limitations in sensitivity or specificity.
Purpose of the Study:
- To develop a novel genetically encoded antibody probe with antigen-dependent fluorescence intensity.
- To enhance the visualization of spatiotemporal dynamics of specific molecules within living cells.
- To create a versatile platform for molecular imaging.
Main Methods:
- Development of a fusion protein: enhanced green fluorescent protein (EGFP) linked to a single-chain variable fragment (scFv) antibody targeting bone Gla protein C-terminus (BGPC7).
- Engineering of the "Flashbody" by inserting circularly permuted GFP into the antibody fragment and optimizing linkers.
- Utilizing live-cell imaging techniques to observe molecular localization and dynamics.
Main Results:
- The BGP Fluobody successfully visualized the intracellular localization of BGPC7-tagged proteins.
- The engineered BGP Flashbody demonstrated a 300% increase in fluorescence intensity upon BGPC7 binding in a dose-dependent manner.
- Live-cell imaging revealed distinct mechanisms for BGPC7 entry through the plasma membrane and nuclear membrane.
Conclusions:
- The Flashbody probe represents a significant advancement in genetically encoded molecular imaging.
- This technology enables enhanced visualization of molecular interactions and dynamics in living cells.
- The Flashbody platform holds potential for imaging a wide range of molecules of interest across various biological studies.
Related Concept Videos
01:12Immunofluorescence Microscopy
14.2K
A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
14.2K
01:37Super-resolution Fluorescence Microscopy
14.7K
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...
14.7K
01:19Protein Dynamics in Living Cells
2.8K
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...
2.8K
01:05Total Internal Reflection Fluorescence Microscopy
13.6K
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
13.6K