What is it about?
Abstract Ultrasensitive detection methods such as laser-induced fluorescence represent the current state-of-the-art in analytics. Single-molecule detection in solution has received a remarkable amount of attention in the last few years because of its applicability to life sciences. Studies have been performed on the fundamentals of the detection processes themselves and on some biological systems. Fluorescence correlation spectroscopy (FCS) is the link for ultrasensitive multicomponent analysis, showing possibilities for experiments on molecular interactions. Based on the theoretical background of FCS, this article gives full explanation of FCS and an update of highlights in experimental biology and medicine studied by FCS. We focus on a repertoire of diverse immunoglobulin specificities, a ribosome display system, single-molecule DNA sequencing, and a mutant enzyme generated by random mutagenesis of amino acids. We describe the usefulness and the enormous potential of the methodology. Further, this contribution clearly Indicates that FCS is a valuable tool for solution-phase single-molecule (SPSM) experiments In immunobiology and medicine. In experiments with the Goodpasture autoantibody, we worked out conditions for the design of experiments on a complex single molecule in solution. The possibility to use SPSM-FCS as a quantitation methodology opens up other important applications beyond the scope of this article. Original results extending the published studies are presented for the rational foundation of SPSM-FCS. In this original contribution, we deal with experimental systems for biology and medicine where the number of molecules In solution is very small. This article Is mandatory for gaining confidence In the interpretation of experimental SPSM-FCS results on the selfsame, Individual single molecule In solution.
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Why is it important?
For theoretical analysis of SPSM-FCS data (measurements) have a deaper look at: https://www.growkudos.com/publications/10.1081%252Fe-emgp-120042041/reader and https://www.growkudos.com/publications/10.14440%252Fjbm.2021.348/reader
Perspectives
See: https://www.nutritionhouse.com/Library/WellnessItem.aspx?ID=74700 and for the novel theory on the time resolution of single molecules in dilute liquids and live cells: https://www.researchgate.net/publication/355034057_Single-molecule_time_resolution_in_dilute_liquids_and_live_cells_at_the_molecular_scale_Constraints_on_the_measurement_time#fullTextFileContent and https://pubmed.ncbi.nlm.nih.gov/33604394/
PRESERVE FROM BEING FORGOTTEN: Professor Zeno Földes-Papp [Biochemist, Gerontologist (Biochemiker, Geriater)]: Laying the Foundation of Single-Molecule Biophysics & Biochemistry Based On the Stochastic Nature of Diffusion: The Individual Molecule, from the Mathematical Core to the Physical Theory. -- I hope that my humble scientific work will be well received by the communities of single-molecule imaging and spectroscopy and by all users of these technologies as well as biotechnologies in the various and different disciplines:
Head of Geriatric Medicine (Medical Director of the Geriatric Service: Sektionsleitung Geriatrie) at Asklepios Klinikum Lindau (Bodensee), Bavaria, Germany
Read the Original
This page is a summary of: A New Dimension for the Development of Fluorescence-Based Assays in Solution: From Physical Principles of FCS Detection to Biological Applications, Experimental Biology and Medicine, May 2002, SAGE Publications,
DOI: 10.1177/153537020222700501.
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Resources
Original research article
Novel theory on the time resolution of single molecules in dilute liquids and live cells: the thercodynamic jitter
Original research article
Formulation of the problem: The Single-molecule level The issue with stochastic thermodynamics is a very high level of abstractness of the probabilistic equations in order to describe the stochastic nature of translational diffusion. Stochastic thermodynamics rules the physical formulation of the single-molecule time-resolution Tm. Let us denote by the equation (1) Tm with respect to the measurement of the same molecule in dilute liquids and live cells without immobilization or significant hydrodynamic flow. Why are the above equations so attractive? There is direct and simple connection with measurements. This connection is based on the diffusion times of molecules. In fact, all conditions are only properties of the stochastic nature of diffusion times of single molecules. In the original papers published in 2006 and 2007 [8,9], where the equations (1) and (2) and the concentration dependence of this modern theory of single-molecule detection at the level of the individual molecule, i.e., one and the same molecule, was formulated for the very first time, the derivations of the equations, remarks and explanations were given to justify the experimental conditions (criteria 1 to 3, the equations (1) and (2) are the criterion 4). In this theory, diffusion times are directly linked to selfsame molecule likelihood estimators as a consequence of the stochastic analysis of the thermodynamic system considered. Further, why is the proportionality factor K of the equation (1) specified by the equation (2)? This is only because of the stochastic nature of diffusion. Of course, these conditions are only technical conditions, which however must be fulfilled in order to apply the equations (1) and (2) to measurements. Thus, the language of stochastic thermodynamics [8,9] is the main advantage of the theory of single-molecule detection at the level of the individual molecule.
Original research article
Single-Phase Single-Molecule Fluorescence Correlation Spectroscopy (SPSM-FCS).
Contributors
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