You might also read
Related Articles
Articles linked to this work by shared authors, journal, and citation graph.
Sort by
Same author
SEM mapping of sequence-specific protein-DNA interactions on long DNA molecules.
Nucleic acids research·2026
You might also read
Articles linked to this work by shared authors, journal, and citation graph.
Updated: Nov 6, 2025
Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
Sangyoon Ko1,2, Jiwoong Kwon1, Sang-Hee Shim1,2
1Center for Molecular Spectroscopy and Dynamics, Institute of Basic Science, Seoul, South Korea.
Researchers developed an improved fluorescent protein called eUnaG that allows for clearer, more accurate imaging of tiny structures inside living cells. By modifying the original protein to be brighter and less prone to clumping, this new tool helps scientists visualize delicate cellular components like vimentin filaments with higher detail and fewer errors.
Area of Science:
Background:
No prior work had resolved the limitations of original fluorescent proteins regarding protein aggregation at high expression levels. Researchers often struggle with labeling artifacts that distort the natural architecture of mammalian cells during imaging. This gap motivated the development of improved protein tags for high-resolution visualization. It was already known that UnaG exhibits reversible fluorescence switching suitable for advanced microscopy. However, the tendency of cytosolic variants to form patches hindered accurate structural assessments. That uncertainty drove the need for a mutant with enhanced brightness and structural stability. Previous studies established the utility of ligand-activatable proteins in single-molecule localization microscopy. Yet, achieving high photon outputs without compromising the endogenous cellular environment remained a significant challenge.
Purpose Of The Study:
The primary aim of this research is to introduce eUnaG as an improved ligand-activatable fluorescent protein for advanced imaging. Scientists sought to address the persistent issue of labeling artifacts that occur during high-level protein expression. The study investigates whether a V2L mutation can enhance brightness while maintaining the reversible switching behavior of the original protein. Researchers intended to demonstrate the utility of this mutant for both conventional and super-resolution microscopy in mammalian cells. The team focused on overcoming the tendency of cytosolic proteins to form aggregated patches. They aimed to provide a more accurate representation of endogenous structures, particularly delicate filaments. This work explores the potential of the new variant to achieve high photon outputs for precise single-molecule localization. The motivation stems from the need for high-performance tools that minimize structural distortion during live-cell imaging.
Main Methods:
The review approach involved evaluating the performance of the V2L mutant against the original protein variant. Investigators utilized mammalian cell lines to test expression levels and structural integrity of labeled targets. They applied blue-light illumination to trigger the photoswitchable properties of the fluorescent tags. Researchers monitored the fluorescence recovery process by introducing specific fluorogenic ligands into the buffer solution. The team performed single-molecule localization microscopy to capture high-resolution images of various subcellular components. They compared the photon count distributions between the two protein versions to determine imaging quality. The study employed sampling coverage analysis to quantify the efficiency of protein labeling on vimentin filaments. Scientists assessed the presence of aggregated patches to identify potential distortions in the cellular architecture.
Main Results:
Key findings from the literature indicate that eUnaG exhibits twice the bulk fluorescence brightness compared to the original protein. The mutant successfully forms proper structures in mammalian cells without the notable distortions seen in previous versions. Specifically, vimentin filaments show significantly better preservation when tagged with this improved variant. The single-molecule photon count distribution remains high, ensuring comparable resolution in super-resolution images. Sampling coverage analysis confirms an improvement in labeling efficiency for the targeted subcellular structures. The protein effectively switches off under blue-light illumination and recovers fluorescence upon ligand addition. Most transiently expressing cells display minimal labeling artifacts at high expression levels. These results establish the tool as a high-performance option for green emission imaging.
Conclusions:
The authors demonstrate that eUnaG serves as a superior protein label for high-resolution imaging applications. Their findings show that this mutant maintains structural integrity in mammalian cells more effectively than its predecessor. The study confirms that vimentin filaments retain their natural organization when tagged with this improved variant. Researchers report that the photon count distribution remains high, supporting excellent image resolution. The analysis of sampling coverage indicates a clear advancement in labeling efficiency for subcellular targets. This work suggests that the new protein minimizes common artifacts associated with high expression levels. The authors conclude that this tool provides a robust solution for green emission imaging requirements. These results offer a reliable method for visualizing delicate biological structures with increased precision.
The researchers propose that eUnaG utilizes a ligand-activatable mechanism where blue-light illumination switches off fluorescence, while high concentrations of fluorogenic ligands restore it. This reversible switching allows for precise control during imaging sessions, unlike standard proteins that remain permanently active.
The authors utilize a V2L mutation, which replaces a specific amino acid to double the bulk fluorescence brightness. This modification distinguishes eUnaG from the original UnaG, providing higher photon outputs necessary for capturing detailed images of cellular components.
The researchers state that high concentrations of fluorogenic ligands are necessary to restore fluorescence after blue-light illumination. This chemical environment ensures that the protein can be toggled repeatedly, which is essential for the localization processes required in super-resolution imaging.
The authors employ sampling coverage analysis to evaluate how well the protein labels target structures. This data type confirms that eUnaG improves labeling efficiency compared to the original version, particularly when visualizing complex networks like vimentin filaments.
The researchers measure the single-molecule photon count distribution to assess image quality. They observe that eUnaG provides a distribution similar to the original protein, which translates into high-resolution images of various subcellular structures without significant loss of detail.
The authors propose that eUnaG is a high-performance tool for green emission imaging because it minimizes labeling artifacts. They claim this improvement allows for more accurate representations of endogenous structures in transiently expressing mammalian cells.
