实验仪器使用技术

Molecular cloning is the process of inserting foreign fragments of DNA into a plasmid or bacteriophage vector that is capable of autonomous replication in a suitable host cell. The resulting recombinant DNA molecules can then be amplified by growth in the host and isolated in pure form. The nucle ...

Defensive behaviors of lower mammals constitute a significant model for understanding human emotional disorders. They generally occur in response to a number of threatening stimuli, including predators, attacking conspecifics, and dangerous objects or situations. Such beh ...

In the last two decades, fluorescent proteins became an indispensable tool to noninvasively label a protein in living cells. The discovery of photoswitchable fluorescent proteins expanded the applications of the fluorescent proteins to techniques such as molecular tracking and h ...

Single-molecule fluorescence studies of nucleic acids are revolutionizing our understanding of fundamental cellular processes related to DNA and RNA processing mechanisms. Detailed molecular insights into DNA repair, replication, transcription, and RNA folding and fun ...

In modern fluorescence fluctuation spectroscopy, the autocorrelation function and photon counting distribution are two widely used statistical characteristics of the measured fluctuating fluorescence intensity signal. Applying special analysis methods such as flu ...

In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlat ...

This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the ...

Dual-color FCS is a powerful method to monitor protein–protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentr ...

Pulsed interleaved excitation (PIE) employs pulsed laser sources that are interleaved such that differentially colored fluorophores can be measured or imaged quasi-simultaneously in the absence of spectral crosstalk. PIE improves the robustness and reduces data analysis co ...

Scanning fluorescence correlation spectroscopy (SFCS) with a scan path perpendicular to the membrane plane was introduced to measure diffusion and interactions of fluorescent components in free-standing biomembranes. Using a confocal laser scanning microscope (CLSM), the ...

Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measu ...

Quenching of organic fluorophores by aromatic amino acids and DNA nucleotides with expelled electron donating properties allows the study of conformational dynamics of biomolecules. Efficient fluorescence quenching via photoinduced electron transfer (PET) requires van ...

Fluorescence correlation spectroscopy (FCS) is a powerful method to investigate molecular interactions based on the variation of diffusion properties at the single-molecule level. This technique allows studying quantitatively the interaction of fluorescently labeled ...

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 d ...

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 ...

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 ...

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-resolv ...

This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of F�rster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protopl ...

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. Here, we present recent achievements in the construction of a positional sensitive wide ...

F�rster 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 b ...