Issue 5, 2018

Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors

Abstract

Single-molecule (sm) fluorescence time series measurements based on the line confocal optical system are a powerful strategy for the investigation of the structure, dynamics, and heterogeneity of biological macromolecules. This method enables the detection of more than several thousands of fluorescence photons per millisecond from single fluorophores, implying that the potential time resolution for measurements of the fluorescence resonance energy transfer (FRET) efficiency is 10 μs. However, the necessity of using imaging photodetectors in the method limits the time resolution in the FRET efficiency measurements to approximately 100 μs. In this investigation, a new photodetector called a hybrid photodetector (HPD) was incorporated into the line confocal system to improve the time resolution without sacrificing the length of the time series detection. Among several settings examined, the system based on a slit width of 10 μm and a high-speed counting device made the best of the features of the line confocal optical system and the HPD. This method achieved a time resolution of 10 μs and an observation time of approximately 5 ms in the sm-FRET time series measurements. The developed device was used for the native state of the B domain of protein A.

Graphical abstract: Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors

Supplementary files

Article information

Article type
Paper
Submitted
22 ذو الحجة 1438
Accepted
14 صفر 1439
First published
14 صفر 1439

Phys. Chem. Chem. Phys., 2018,20, 3277-3285

Microsecond resolved single-molecule FRET time series measurements based on the line confocal optical system combined with hybrid photodetectors

H. Oikawa, T. Takahashi, S. Kamonprasertsuk and S. Takahashi, Phys. Chem. Chem. Phys., 2018, 20, 3277 DOI: 10.1039/C7CP06268K

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements