Fluorescence Fluctuation Spectroscopy Techniques To Quantify Molecular Interactions And Dynamics In Complex Biological Systems

Living cells rely on transport and interaction of biomolecules to perform their diverse functions. A powerful toolbox to study these highly dynamic processes in the native environment is provided by fluorescence fluctuation spectroscopy (FFS) techniques. In more detail, FFS takes advantage of the inherent dynamics present in biological systems, such as diffusion, to infer molecular parameters from fluctuations of the signal emitted by an ensemble of fluorescently tagged molecules. In particular, two parameters are accessible: the concentration of molecules and their transit times through the observation volume. In addition, molecular interactions can be measured by analyzing the average signal emitted per molecule - the molecular brightness - and the cross-correlation of signals detected from differently tagged species. In the present work, several FFS techniques were implemented and applied in different biological contexts. In particular, scanning fluorescence correlation spectroscopy (sFCS) was performed to measure protein dynamics ...

This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. This volume covers Fluorescence Fluctuation Spectroscopy Contains chapters on such topics as Time-integrated fluorescence cumulant analysis, Pulsed Interleaved Excitation, and raster image correlation spectroscopy and number and brightness analysis. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers fluorescence fluctuation spectroscopy Contains chapters on such topics as time-integrated fluorescence cumulant analysis, pulsed interleaved excitation, and raster image correlation spectroscopy and number and brightness analysis.

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers fluorescence fluctuation spectroscopy Contains chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells

Biomolecules such as proteins and lipids have vital roles in numerous cellular functions, including biomolecule transport, protein functions, cellular homeostasis and biomembrane integrity. Traditional biochemistry methods do not provide precise information about cellular biomolecule distribution and behavior under native environmental conditions since they are not transferable to live cell samples. Consequently, this can lead to inaccuracies in quantifying biomolecule interactions due to potential complexities arising from the heterogeneity of native biomembranes. To overcome these limitations, minimal invasive microscopic techniques, such as fluorescence fluctuation spectroscopy (FFS) in combination with fluorescence proteins (FPs) and fluorescence lipid analogs, have been developed. FFS techniques and membrane property sensors enable the quantification of various parameters, including concentration, dynamics, oligomerization, and interaction of biomolecules in live cell samples. In this work, several FFS approaches and membrane ...

Fluorescence microscopy has proven to be immensely powerful for the study of biological systems at both the cellular and systems biological levels. The ability to specifically label a single molecular species fluorescently has enabled the study of complex cellular structures through the visualization of their constituent components both individually as well as in context of the overall structure. Since the advent of engineered fluorescent proteins (such as GFP) and other proteins capable of being genetically encoded as fusion constructs, the utility of fluorescence microscopy has increased exponentially in terms of the ability to efficiently, specifically label desired molecules while limiting perturbations to the biology under study. With this enhanced ability of visualization came a hand-in-hand evolution of computational techniques to extract quantitative information from microscopy images. In this thesis, I focus on the application of fluorescence imaging at the biophysical level in living cells: analyzing the motion/dynamics of single molecules and complexes, which are small relative to the structures of the cell, in order to elucidate their molecular function and mechanism. The motion of these "particles" within living cells is necessarily related to their functions as well as their interacting partners, which can vary dynamically during their lifetimes. Observation and analysis of this motion using a combination of fluorescence microscopy and robust quantitative analysis allows one to infer these characteristics. Here, I study three diverse biological systems in the context of live-cell fluorescence microscopy and biophysical analysis: 1) the transport of 0-actin mRNA particles in primary mouse neurons, 2) kinetochore motion during cell division, specifically focusing on anaphase dynamics, and 3) the motion of cell-growth-implicated membrane proteins in Bacillus subtilis.

This is the first book-length treatment of both the theoretical background to fluorescence correlation spectroscopy (FCS) and a variety of applications in various fields of science. The high spatial and temporal resolution of FCS has made it a powerful tool for the analysis of molecular interactions and kinetics, transport properties due to thermal motion, and flow. It contains an essential contribution from Nobel Prize winner M. Eigen, who is credited with inventing FCS.

Volume 3 of this new series focuses on brandnew research and applications in biology, biophysics and other fields of life sciences. Many frontline researcher have contributed to this highly attractive and interdisciplinary volume which spans the entire field of present fluorescence spectroscopy including nanotechnology, membrane and DNA studies and fluorescence imaging in cancer research.

The key element of any fluorescence sensing or imaging technology is the fluorescence reporter, which transforms the information on molecular interactions and dynamics into measurable signals of fluorescence emission. This book, written by a team of frontline researchers, demonstrates the broad field of applications of fluorescence reporters, starting from nanoscopic properties of materials, such as self-assembled thin films, polymers and ionic liquids, through biological macromolecules and further to living cell, tissue and body imaging. Basic information on obtaining and interpreting experimental data is presented and recent progress in these practically important areas is highlighted. The book is addressed to a broad interdisciplinary audience.

Fluorescence spectroscopy is a type of electromagnetic spectroscopy, using a beam of light, which analyzes fluorescence from a sample. Given its extremely high sensitivity and selectivity, it is an important investigational tool in many areas including material sciences, analytical sciences, and across a broad range of chemical, biochemical and medical research. It has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems. The pharmaceutical industry uses it heavily and it has become a dominating technique in biochemistry and molecular genetics. Keeps MIE buyers and online subscribers up-to-date with the latest research with this highly used technique Provides tried and tested techniques which eliminate searching through many different sources

This is the first book-length treatment of both the theoretical background to fluorescence correlation spectroscopy (FCS) and a variety of applications in various fields of science. The high spatial and temporal resolution of FCS has made it a powerful tool for the analysis of molecular interactions and kinetics, transport properties due to thermal motion, and flow. It contains an essential contribution from Nobel Prize winner M. Eigen, who is credited with inventing FCS.