scholarly journals I. Conformational Dynamics of Biological Macromolecules by Polarization-Modulated Fourier Imaging Correlation Spectroscopy

2009 ◽  
Vol 113 (19) ◽  
pp. 6847-6853 ◽  
Author(s):  
Geoffrey A. Lott ◽  
Eric N. Senning ◽  
Michael C. Fink ◽  
Andrew H. Marcus
Keyword(s):  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Biological Macromolecules ◽  
Fourier Imaging ◽  
Fourier Imaging Correlation Spectroscopy

scholarly journals EXPRESS: Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

2021 ◽  
pp. 000370282110099
Author(s):  
Ziyu Yang ◽  
Haiqi Xu ◽  
Jiayu Wang ◽  
Wei Chen ◽  
Meiping Zhao
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Membrane Receptors ◽  
Biological Macromolecules ◽  
Dynamics Analysis ◽  
Single Molecule Fluorescence ◽  
Membrane Protein Dynamics

Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

scholarly journals Fast and Robust 2D Inverse Laplace Transformation of Single-Molecule Fluorescence Lifetime Data

2021 ◽  
Author(s):  
Saurabh Talele ◽  
John T. King
Keyword(s):  
Nmr Spectroscopy ◽  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Great Promise ◽  
Biological Macromolecules ◽  
Single Molecule Fluorescence ◽  
Laplace Transformations

AbstractFluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule 2D-fluorescence lifetime correlation spectroscopy (sm-2D-FLCS) is an emerging technique that holds great promise for the study of protein and nucleic acid dynamics as it 1) resolves conformational dynamics using a single chromophore, 2) measures forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transition (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. The current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform 1D and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data.Significance StatementInverse Laplace transformations are a powerful approach for analyzing relaxation data. The inversion computes a relaxation rate spectrum from experimentally measured temporal relaxation, circumventing the need to choose appropriate fitting functions. They are routinely performed in NMR spectroscopy and are becoming increasing used in single-molecule fluorescence experiments. However, as Laplace inversions are ill-conditioned transformations, they must be estimated from regularization algorithms that are often computationally costly and difficult to implement. In this work, we adopt an algorithm first developed for NMR relaxometry to provide fast, robust, and easy to implement 1D and 2D inverse Laplace transformations on single-molecule fluorescence data.

scholarly journals II. Kinetic Pathways of Switching Optical Conformations in DsRed by 2D Fourier Imaging Correlation Spectroscopy

2009 ◽  
Vol 113 (19) ◽  
pp. 6854-6860 ◽  
Author(s):  
Eric N. Senning ◽  
Geoffrey A. Lott ◽  
Michael C. Fink ◽  
Andrew H. Marcus
Keyword(s):  
Correlation Spectroscopy ◽  
Fourier Imaging ◽  
Fourier Imaging Correlation Spectroscopy
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