Imaging of Plant Materials Using Indirect Desorption Electrospray Ionization Mass Spectrometry
Indirect desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a method for imaging distributions of metabolites in plant materials, in particular leaves and petals. The challenge in direct imaging of such plant materials with DESI-MS is particularly the protective layer of cuticular wax present in leaves and petals. The cuticle protects the plant from drying out, but also makes it difficult for the DESI sprayer to reach the analytes of interest inside the plant material. A solution to this problem is to imprint the plant material onto a surface, thus releasing the analytes of interest from parts of the...
Source: Springer protocols feed by Imaging/Radiology - November 3, 2014 Category: Radiology Source Type: news

Desorption Electrospray Ionization Imaging of Small Organics on Mineral Surfaces
Desorption electrospray ionization (DESI)-mass spectrometry facilitates the ambient chemical analysis of a variety of surfaces. Here we describe the protocol for using DESI imaging to measure the distributions of small prebiotically relevant molecules on granite surfaces. Granites that contain a variety of juxtaposed mineral species were reacted with formamide in order to study the role of local mineral environment on the production of purines and pyrimidines. The mass spectrometry imaging (MSI) methods described here can also be applied to the surface analysis of rock samples involved in other applications such as petrole...
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DESI Imaging of Small Molecules in Biological Tissues
Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or preprocessed samples under ambient conditions. Among other applications, DESI is used to identify and to record spatial distributions of small molecules in situ, sliced or imprinted biological tissue. Manipulation of the chemistry accompanying ambient analysis ionization can be used to optimize chemical analysis, including molecular imprinting. Images are obtained by continuously moving the sample relative to the DESI sprayer and the inlet of the mass spectrometer. The acquisition time depends on the size of the surface to be analyz...
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Multiplex MALDI-MS Imaging of Plant Metabolites Using a Hybrid MS System
Plant tissues present intriguing systems for study by mass spectrometry imaging, as they exhibit a complex metabolism and a high degree of spatial localization. This chapter presents a methodology for preparation of plant tissue sections for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) analysis and the use of a hybrid mass spectrometer for “multiplex” imaging. The multiplex method described here provides a wide range of analytical information, including high-resolution, accurate mass imaging and tandem MS scans for structural information, all within a single experiment. Whil...
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MALDI Mass Spectrometry Imaging of Lipids and Primary Metabolites on Rat Brain Sections
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) enables the localization and the structural identification of a large set of molecules on a surface with 10–20 μm resolution. MALDI is often coupled to time-of-flight (TOF) tandem analyzers which remain a versatile instrument for the detection and the structural analysis of molecular ions. This technique can be used to locate either large biomolecules, such as peptides/proteins, or small endogenous or exogenous ones. Among them, lipids and primary metabolites are of high interest because they can reflect the cell state. This chapter ...
Source: Springer protocols feed by Imaging/Radiology - November 3, 2014 Category: Radiology Source Type: news

MALDI-MS-Assisted Molecular Imaging of Metabolites in Legume Plants
Mass spectrometric imaging (MSI) is a powerful analytical tool that provides spatial information of several compounds in a single experiment. This technique has been used extensively to study proteins, peptides, and lipids, and is becoming more common for studying small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected within a biological tissue section. Herein, we present a method developed specifically for imaging metabolites in legume plant roots and root nodules which can be adapted for stu...
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TOF-SIMS Imaging of Lipids on Rat Brain Sections
Since several decades, secondary ion mass spectrometry (SIMS) coupled to time of flight (TOF) is used for atomic or small inorganic/organic fragments imaging on different materials. With the advent of polyatomic ion sources leading to a significant increase of sensitivity in combination with a reasonable spatial resolution (1–10 μm), TOF-SIMS is becoming a more and more popular analytical platform for MS imaging. Even if this technique is limited to small molecules (typically below 1,000 Da), it offers enough sensitivity to detect and locate various classes of lipids directly on the surface of tissue sections. Thi...
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Sample Preparation for 3D SIMS Chemical Imaging of Cells
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is an emerging technique for the characterization of biological systems. With the development of novel ion sources such as cluster ion beams, ionization efficiency has been increased, allowing for greater amounts of information to be obtained from the sample of interest. This enables the plotting of the distribution of chemical compounds against position with submicrometer resolution, yielding a chemical map of the material. In addition, by combining imaging with molecular depth profiling, a complete 3-dimensional rendering of the object is possible. The study of si...
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Current Status and Future Prospects of Mass Spectrometry Imaging of Small Molecules
In the field of small-molecule studies, vast efforts have been put forth in order to comprehensively characterize and quantify metabolites formed from complex mechanistic pathways within biochemical and biological organisms. Many technologies and methodologies have been developed to aid understanding of the inherent complexities within biological metabolomes. Specifically, mass spectroscopy imaging (MSI) has emerged as a foundational technique in gaining insight into the molecular entities within cells, tissues, and whole-body samples. In this chapter we provide a brief overview of major technical components involved in MS...
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Data Processing and Analysis for Mass Spectrometry Imaging
Mass spectrometry imaging produces large numbers of spectra that need to be efficiently stored, processed, and analyzed. In this chapter, we describe the protocol and methods for data processing, visualization, and statistical analysis, with related techniques and tools available presented. Examples are given with data collected for a 3D MS imaging of a mouse brain and 2D MS imaging of human bladder tissues. (Source: Springer protocols feed by Imaging/Radiology)
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Laser Desorption Postionization Mass Spectrometry Imaging of Biological Targets
Laser desorption photoionization mass spectrometry (LDPI-MS) utilizes two separate light sources for desorption and photoionization of species from a solid surface. This technique has been applied to study a wide variety of molecular analytes in biological systems, but is not yet available in commercial instruments. For this reason, a generalized protocol is presented here for the use of LDPI-MS imaging to detect small molecules within intact biological samples. Examples are provided here for LDPI-MS imaging of an antibiotic within a tooth root canal and a metabolite within a coculture bacterial biofilm. (Source: Springer ...
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Matrix-Enhanced Surface-Assisted Laser Desorption/Ionization Mass Spectrometry (ME-SALDI-MS) for Mass Spectrometry Imaging of Small Molecules
We describe herein a detailed protocol that utilizes a hybrid LDI method, matrix-enhanced SALDI-MS (ME-SALDI MS), to detect and image low MW species in an imaging mode. (Source: Springer protocols feed by Imaging/Radiology)
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Nanoparticle-Assisted Laser Desorption/Ionization for Metabolite Imaging
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has enabled the spatial analysis of various molecules, including peptides, nucleic acids, lipids, and drug molecules. To expand the capabilities of MALDI-IMS, we have established an imaging technique using metal nanoparticles (NPs) to visualize metabolites, termed nanoparticle-assisted laser desorption/ionization imaging mass spectrometry (nano-PALDI-IMS). By utilizing Ag-, Fe-, Au-, and TiO2-derived NPs, we have succeeded in visualizing various metabolites, including fatty acid and glycosphingolipids, with higher sensitivity and spatial reso...
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Nanostructure-Initiator Mass Spectrometry (NIMS) for Molecular Mapping of Animal Tissues
We describe here a simple step-by-step protocol for substrate fabrication and sample preparation that provides a starting point for the technique to be adapted and optimized for 2-D biological imaging applications. (Source: Springer protocols feed by Imaging/Radiology)
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Nanostructure Imaging Mass Spectrometry: The Role of Fluorocarbons in Metabolite Analysis and Yoctomole Level Sensitivity
Nanostructure imaging mass spectrometry (NIMS) has become an effective technology for generating ions in the gas phase, providing high sensitivity and imaging capabilities for small molecules, metabolites, drugs, and drug metabolites. Specifically, laser desorption from the nanostructure surfaces results in efficient energy transfer, low background chemical noise, and the nondestructive release of analyte ions into the gas phase. The modification of nanostructured surfaces with fluorous compounds, either covalent or non-covalent, has played an important role in gaining high efficiency/sensitivity by facilitating analyte de...
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Laser Ablation Sample Transfer for Mass Spectrometry Imaging
Infrared laser ablation sample transfer (IR-LAST) is a novel ambient sampling technique for mass spectrometry. In this technique, a pulsed mid-IR laser is used to ablate materials that are collected for mass spectrometry analysis; the material can be a solid sample or deposited on a sample target. After collection, the sample can be further separated or analyzed directly by mass spectrometry. For IR-LAST sample transfer tissue imaging using MALDI mass spectrometry, a tissue section is placed on a sample slide and material transferred to a target slide by scanning the tissue sample under a focused laser beam using transmiss...
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Automated Cell-by-Cell Tissue Imaging and Single-Cell Analysis for Targeted Morphologies by Laser Ablation Electrospray Ionization Mass Spectrometry
Mass spectrometry imaging (MSI) is an emerging technology for the mapping of molecular distributions in tissues. In most of the existing studies, imaging is performed by sampling on a predefined rectangular grid that does not reflect the natural cellular pattern of the tissue. Delivering laser pulses by a sharpened optical fiber in laser ablation electrospray ionization (LAESI) mass spectrometry (MS) has enabled the direct analysis of single cells and subcellular compartments. Cell-by-cell imaging had been demonstrated using LAESI-MS, where individual cells were manually selected to serve as natural pixels for tissue imagi...
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Electrospray Laser Desorption Ionization (ELDI) Mass Spectrometry for Molecular Imaging of Small Molecules on Tissues
The use of an ambient ionization mass spectrometry technique known as electrospray laser desorption ionization mass spectrometry (ELDI/MS) for molecular imaging is described in this section. The technique requires little or no sample pretreatment and the application of matrix on sample surfaces is unnecessary. In addition, the technique is highly suitable for the analysis of hard and thick tissues compared to other molecular imaging methods because it does not require production of thin tissue slices via microtomes, which greatly simplifies the overall sample preparation procedure and prevents the redistribution of analyte...
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Imaging of Lipids and Metabolites Using Nanospray Desorption Electrospray Ionization Mass Spectrometry
Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that uses localized liquid extraction for mass spectrometry imaging of molecules on surfaces. Nano-DESI enables imaging of ionizable molecules from a sample in its native state without any special sample pretreatment. In this chapter we describe the protocol for nano-DESI imaging of thin tissue sections. (Source: Springer protocols feed by Imaging/Radiology)
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Fluorescence Cross-Correlation Spectroscopy (FCCS) in Living Cells
Fluorescence cross-correlation spectroscopy (FCCS) is a single-molecule sensitive technique to quantitatively study interactions among fluorescently tagged biomolecules. Besides the initial implementation as dual-color FCCS (DC-FCCS), FCCS has several powerful derivatives, including single-wavelength FCCS (SW-FCCS), two-photon FCCS (TP-FCCS), and pulsed interleaved excitation FCCS (PIE-FCCS). However, to apply FCCS successfully, one needs to be familiar with procedures ranging from fluorescent labeling, instrumentation setup and alignment, sample preparation, and data analysis. Here, we describe the procedures to apply FCC...
Source: Springer protocols feed by Imaging/Radiology - October 14, 2013 Category: Radiology Source Type: news

Application of Fluorescence Correlation Spectroscopy (FCS) to Measure the Dynamics of Fluorescent Proteins in Living Cells
Fluorescence correlation spectroscopy (FCS) can add dynamic molecular information to images of live cells. For example, a confocal laser scanning microscope (CLSM) equipped with an accessory FCS unit provides the possibility to first image the spatial distribution of a fluorescent protein before probing its mobility within defined regions of interest. Whereas specific protein–protein interactions are preferably assayed with a dual-color approach, single-color FCS can still provide valuable information about the size of the diffusing entities and potential interactions with other, nonfluorescent, proteins or subcellul...
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Multimodal Fluorescence Imaging Spectroscopy
Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample. This chapter describes how to build instrumentation that allows for multimodal fluorescence imaging and explains data analysis procedures for the observables. (Source: Springer protocols feed by Imaging/Radiology)
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Time-Resolved Fluorescence Anisotropy Imaging
Fluorescence can be characterized by its intensity, position, wavelength, lifetime, and polarization. The more of these features are acquired in a single measurement, the more can be learned about the sample, i.e., the microenvironment of the fluorescence probe. Polarization-resolved fluorescence lifetime imaging—time-resolved fluorescence anisotropy imaging, TR-FAIM—allows mapping of viscosity or binding or of homo-FRET which can indicate dimerization or generally oligomerization. (Source: Springer protocols feed by Imaging/Radiology)
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Global Analysis of FRET–FLIM Data in Live Plant Cells
This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software. Using this procedure it is possible to ex...
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Wide-Field Fluorescence Lifetime Imaging with Multi-anode Detectors
Fluorescence lifetime imaging microscopy (FLIM) has become a powerful and widely used tool to monitor inter- and intramolecular dynamics of fluorophore-labeled proteins inside living cells. (Source: Springer protocols feed by Imaging/Radiology)
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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...
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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...
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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...
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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 ...
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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)
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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...
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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...
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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...
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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...
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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)
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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 ...
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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)
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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...
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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)
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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 ...
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High-Pressure Fluorescence Applications
Fluorescence is the most widely used technique to study the effect of pressure on biochemical systems. The use of pressure as a physical variable sheds light into volumetric characteristics of reactions. Here we focus on the effect of pressure on protein solutions using a simple unfolding example in order to illustrate the applications of the methodology. Topics covered in this review include the relationships between practical aspects and technical limitations; the effect of pressure and the study of protein cavities; the interpretation of thermodynamic and relaxation kinetics; and the study of relaxation amplitudes. Fina...
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Quantitative Fluorescence Spectral Analysis of Protein Denaturation
This chapter describes a procedure of global analysis of the steady-state spectra measured with different concentrations of the denaturant to quantitatively study protein denaturation. With the help of physicochemical models, relevant spectral parameters that characterize the folding intermediate and thermodynamic parameters that describe a three-state model N  $$ \Leftrightarrow $$  I  $$ \Leftrightarrow $$  U can be estimated. (Source: Springer protocols feed by Imaging/Radiology)
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Steady-State Fluorescence Polarization/Anisotropy for the Study of Protein Interactions
Fluorescence methods are often employed for the characterization of molecular interactions. In particular, polarization/anisotropy studies are widely utilized in the life sciences as they allow quantification of protein interactions in the micro- and nanomolar concentration range. Herein we shall briefly describe the theoretical aspects of polarization/anisotropy and outline an experiment for determination of the dissociation constant for a protein–ligand complex. (Source: Springer protocols feed by Imaging/Radiology)
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How to Collect National Institute of Standards and Technology (NIST) Traceable Fluorescence Excitation and Emission Spectra
Contemporary spectrofluorimeters comprise exciting light sources, excitation and emission monochromators, and detectors that without correction yield data not conforming to an ideal spectral response. The correction of the spectral properties of the exciting and emission light paths first requires calibration of the wavelength and spectral accuracy. The exciting beam path can be corrected up to the sample position using a spectrally corrected reference detection system. The corrected reference response accounts for both the spectral intensity and drift of the exciting light source relative to emission and/or transmission d...
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