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Measuring photoactivation efficiency at the single molecule level

Tue, 01/07/2014 - 8:55am

Human glycine receptor used as a nanotemplate by attaching fluorescent proteins to these subunits to measure photoactivation efficiency at the single molecule level.In a recent study carried out at The Institute of Photonic Sciences (ICFO) and published in Nature Methods, the advanced fluorescence imaging and biophysics group led by Nest Fellow Dr. Melike Lakadamyali was able to quantify the photoactivation efficiency of all the known "irreversibly photoswitching fluorescent proteins" and establish a proper detailed reference framework for determining protein stoichiometry.

The ability to determine protein stoichiometry and monitor changes in the balance between monomeric, dimeric and multimeric proteins allows scientists to see the difference between a properly functioning cell and a diseased cell. For this reason, there is a great interest in being able to count proteins and determine their stoichiometry.

Thanks to the discovery of photoactivatable fluorescent proteins and the development of super-resolution microscopy, “molecular counting” is close to becoming a reality. Photoactivatable flourescent proteins change their fluorescence property from dark to bright when exposed to light. Through the use of localization-based super-resolution microscopy, researchers are able to photoactivate, image, and follow these genetically encoded fluorescent proteins one at a time to study what is happening inside a cell at the molecular level.

However, despite the capability for “molecular counting” that seems intrinsic to the imaging strategy (activating one fluorescent protein at a time should also permit the counting of total existing fluorescent proteins) relating the number of counted fluorescent proteins to actualprotein stoichiometry has been difficult. One important reason for this difficulty has been that, until recently, it was not known whether all fluorescent proteins become bright when exposed to laser light. Failure to photoactivate would lead to undercounting since a fraction of probes would be dark and never appear in the image.

In a recent study carried out at The Institute of Photonic Sciences (ICFO) and published in Nature Methods, the advanced fluorescence imaging and biophysics group led by Nest Fellow Dr. Melike Lakadamyali was able to quantify the photoactivation efficiency of all the known "irreversibly photoswitching fluorescent proteins" and establish a proper detailed reference framework for determining protein stoichiometry. To do this, they used a nanotemplate of known stoichiometry and studied several fluorescent proteins to see the percentage of proteins that was photoactivated. Taking into account the measured photoactivation efficiency as well as other photophysical factors such as blinking, the group was able to determine which proteins are best suited for molecular counting. They found that most fluorescent proteins fail to photoactivate around 50% of the time, thus underscoring the importance of accounting for the photoactivation efficiency to correctly interpret the quantitative information.

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Research Group led by Prof. Melike Lakadamyali

Source: The Institute of Photonic Sciences

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