Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

Fluorescence spectrometry is the most extensively used optical spectroscopic method in analytical measurement and scientific investigation. During the past five years more than 60000 scientific articles have been published in which fluorescence spectroscopy has been used. The large number of applications ranges from the analytical determination of trace metals in the environment to pH measurements in whole cells under physiological conditions. In the scientific research laboratory, fluorescence spectroscopy is being used or applied to study the fundamental physical processes of molecules; structure-function relationships and interactions of biomolecules such as proteins and nucleic acids; structures and activity within whole cells using such instrumentation as confocal microscopy; and DNA sequencing in genomic characterization. In analytical applications the use of fluorescence is dominant in clinical laboratories where fluorescence immunoassays have largely replaced radioimmunoassay techniques. There are two main reasons for this extensive use of fluorescence spectroscopy. Foremost is the high level of sensitivity and wide dynamic range that can be achieved. There are a large number of laboratories that have reported single molecule detection. Secondly, the instrumentation required is convenient and for most purposes can be purchased at a modest cost. While improvements and advances continue to be reported fluorescence instrumentation has reached a high level of maturity. A review of the physical principles of the fluorescence phenomenon permits one to understand the origins of the information content that fluorescence measurements can provide. A molecule absorbs electromagnetic radiation through a quantum mechanical process where the molecule is transformed from a ‘ground’ state to an ‘excited’ state. The energy of the absorbed photon of light corresponds to the energy difference between these two states. In the case of light in the ultraviolet and visible spectral range of 200 nm to 800 nm that corresponds to energies of 143 to 35.8 kcal mol-1. The absorption of light results in an electronic transition in the atom or molecule. In atoms this involves the promotion of an electron from an outer shell orbital to an empty orbital of higher energy.

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Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

10.1007/978-1-0716-1716-8_12 ◽

2021 ◽

pp. 207-233

Author(s):

Franziska Zosel ◽

Andrea Holla ◽

Benjamin Schuler

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Single Molecule Fluorescence ◽

Single Molecule Fluorescence Spectroscopy

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Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Molecules ◽

10.3390/molecules24152826 ◽

2019 ◽

Vol 24 (15) ◽

pp. 2826 ◽

Cited By ~ 4

Author(s):

Shangguo Hou ◽

Courtney Johnson ◽

Kevin Welsher

Keyword(s):

Fluorescence Spectroscopy ◽

Real Time ◽

Temporal Resolution ◽

Single Molecule ◽

Particle Tracking ◽

Single Particle ◽

Single Particle Tracking ◽

Single Molecule Fluorescence ◽

Single Molecule Fluorescence Spectroscopy ◽

Active Feedback

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

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