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A Quantitative Protocol for Intensity-Based Live Cell FRET Imaging
Förster resonance energy transfer (FRET) has become one of the most ubiquitous and powerful methods to quantify protein interactions in molecular biology. FRET refers to the sensitization of an acceptor molecule through transfer of energy from a nearby donor, and it can occur if the emission band of the donor exhibits spectral overlap with the absorption band of the acceptor molecule. Numerous methods exist to quantify FRET levels from interacting protein labels including fluorescence lifetime, acceptor photobleaching, and polarization-resolved imaging (Lakowicz, Principles of fluorescence spectroscopy, 2006; Jares-Er...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Rectangle FRAP for Measuring Diffusion with a Laser Scanning Microscope
Fluorescence recovery after photobleaching (FRAP) is one of the most useful microscopy techniques for studying the mobility of molecules in terms of a diffusion coefficient. Here, we describe a FRAP method that allows such measurements, relying on the photobleaching of a rectangular region of any size and aspect ratio. We start with a brief overview of the rectangle FRAP theory, and next we provide guidelines for performing FRAP measurements, including a discussion of the experimental setup and the data analysis. Finally, we discuss how to verify correct use of the rectangle FRAP method using test solutions. (Source: Sprin...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Monitoring Membrane Properties and Apoptosis Using Membrane Probes of the 3-Hydroxyflavone Family
Environment-sensitive fluorescent membrane probes are attractive tools for investigating the membrane properties and their changes under perturbing conditions. Membrane probes of the 3-hydroxyflavone family are of particular interest due to their excited-state intramolecular proton transfer (ESIPT) reaction, which confers a dual emission highly sensitive to the polarity and hydration of the environment. In the present work, we will describe the protocols used with these probes in order to monitor the physicochemical properties of lipid membrane models and cell plasma membranes and to detect apoptosis. (Source: Springer pro...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Optimization of Fluorescent Proteins
Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and microcopy techniques. This has allowed the (in situ) study of biomolecules with unprecedented resolution, specificity, sensitivity, and ease of labeling. However, brighter FPs, more photostable FPs, and FPs that display an even better compatibility with biophysical microspectroscopic techniques are still highly desired. The key characteristics of ...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Biosynthetic Incorporation of Tryptophan Analogs in Proteins
Biosynthetic incorporation of Trp analogs in a protein can help in its characterization using fluorescence spectroscopy and other methodologies like NMR and phosphorescence. Here a protocol is presented resulting in the efficient incorporation of Trp analogs in a recombinant protein, using an Escherichia coli Trp auxotroph. An overview of recent developments in the Trp analog incorporation field is also presented. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Analysis of Time-Dependent Red Shifts in Fluorescence Emission from Tryptophan Residues in Proteins
Instantaneous fluorescence emission spectra measured at different times after excitation often shift to the red as the delay between the excitation pulse and fluorescence detection is increased. In the case of Trp fluorescence in proteins, the time-dependent red shift (TDRS) may have its origins in relaxation, heterogeneity, or a mixture of the two. In those cases where it is possible to rule out the contribution of heterogeneity, the TDRS can be used to study nonequilibrium relaxation dynamics of the protein matrix and the solvent on the picosecond and nanosecond time scales. Here we describe the experimental and computat...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

MD + QM Correlations with Tryptophan Fluorescence Spectral Shifts and Lifetimes
Principles behind quenching of tryptophan (Trp) fluorescence are updated and extended in light of recent 100-ns and 1-μs molecular dynamics (MD) trajectories augmented with quantum mechanical (QM) calculations that consider electrostatic contributions to wavelength shifts and quenching. Four studies are summarized, including (1) new insight into the single exponential decay of NATA, (2) a study revealing how unsuspected rotamer transitions affect quenching of Trp when used as a probe of protein folding, (3) advances in understanding the origin of nonexponential decay from 100-ns simulations on 19 Trps in 16 proteins, an...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Ensemble and Single-Molecule Detected Time-Resolved FRET Methods in Studies of Protein Conformations and Dynamics
Most proteins are nanomachines that are selected to execute specific functions and therefore should have some degree of flexibility. The driving force that excites specific motions of domains and smaller chain elements is the thermal fluctuations of the solvent bath which are channeled to selected modes of motions by the structural constraints. Consequently characterization of the ensembles of conformers of proteins and their dynamics should be expressed in statistical terms, i.e., determination of probability distributions of the various conformers. This can be achieved by measurements of time-resolved dynamic non-radiati...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Polar Plot Representation of Time-Resolved Fluorescence
Measuring changes in a molecule’s fluorescence emission is a common technique to study complex biological systems such as cells and tissues. Although the steady-state fluorescence intensity is frequently used, measuring the average amount of time that a molecule spends in the excited state (the fluorescence lifetime) reveals more detailed information about its local environment. The lifetime is measured in the time domain by detecting directly the decay of fluorescence following excitation by short pulse of light. The lifetime can also be measured in the frequency domain by recording the phase and amplitude of oscill...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Frequency Domain Fluorometry: Theory and Application
Frequency domain fluorometry is a widely utilized tool in the physical, chemical, and biological sciences. This chapter focuses on the theory of the method and the practical aspects required to carry out intensity decay, i.e., lifetime measurements on a modern frequency domain fluorometer. Several chemical/biological systems are utilized to illustrate data acquisition protocols. Data analysis procedures and methodologies are also discussed. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Photoinduced Electron Transfer Modeling to Simulate Flavoprotein Fluorescence Decay
A method of analysis is described on the photoinduced electron transfer (PET) from aromatic amino acids as tryptophans (Trp) and tyrosines (Tyr) to the excited isoalloxazine (Iso*) in FMN-binding proteins (FBP) from Desulfovibrio vulgaris (strain, Miyazaki F). Time-dependent geometrical factors as the donor–acceptor distances are determined by means of a molecular dynamics simulation (MDS) of the proteins. Fluorescence decays of the single mutated isoforms of FBP are used as experimental data. The electrostatic (ES) energy between the photoproducts and ionic groups in the proteins is introduced into the Kakitani and ...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Subpicosecond Kerr-Gate Spectrofluorometry
This chapter describes an experimental layout for time and spectrally resolved fluorescence measurements with femtosecond time resolution based on Kerr gating. The combination of data recorded using different Kerr media allows a temporal dynamic range from ~100 fs to several nanoseconds. Simultaneous analysis of multiple datasets is described. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Upconversion Spectrophotofluorometry
As the other chapters attest, sensitivity of fluorescent molecules to their local environment has created powerful tools in the study of molecular biology, particularly in the study of protein, DNA, and lipid dynamics. Surprisingly, even events faster than the nanosecond lifetimes of fluorophores are important in protein function, and in particular, events lasting just a few ps reflect on water motion and the coupled dynamics of proteins. These ultrafast phenomena can best be studied by using the same laser that excites fluorescence to also “strobe” the emission, providing sub-picosecond time slices of the acti...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Nanometrology
Methods and protocols are described when using fluorescence metrology to determine the average nanoparticle (np) size in colloids in the range of 1–10 nm. The technique is based on determining the rotational correlation time of the np from the decay of fluorescence anisotropy of a dye that is electrostatically or covalently attached to the np as it undergoes Brownian rotation. The np size is then calculated from the Stokes–Einstein equation. The exemplar of silica nps is presented, but the approach can also be applied to other types of nps. (Source: Springer protocols feed by Imaging/Radiology)
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Global Analysis of Time-Resolved Fluorescence Data
In this chapter, we describe the global analysis approach for processing time-resolved fluorescence spectroscopy data of molecules in the condensed phase. Combining simultaneous analysis of data measured under different experimental conditions (spatial coordinates, temperature, concentration, emission wavelength, excitation intensity, etc.) with the fitting strategy, enabling parameter linkage and thus decreasing the total amount of estimated variables, makes global analysis more robust and more consistent compared to a sequential fit of single experimental data. We consider the main stages of the global analysis approach ...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news