An introduction to the fluorescence study of the kinetics of energy

This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. Simple statistical mechanical models have been extremely useful in interpreting the experimental results.

An introduction to the fluorescence study of the kinetics of energy

Notice that the fraction is the reciprocal of that used for lifetime measurements. This technique was introduced by Jovin in Also, the fact that time measurements are over seconds rather than nanoseconds makes it easier than fluorescence lifetime measurements, and because photobleaching decay rates do not generally depend on donor concentration unless acceptor saturation is an issuethe careful control of concentrations needed for intensity measurements is not needed.

Related JoVE Videos

It is, however, important to keep the illumination the same for the with- and without-acceptor measurements, as photobleaching increases markedly with more intense incident light. Lifetime measurements[ edit ] FRET efficiency can also be determined from the change in the fluorescence lifetime of the donor.

Lifetime measurements of FRET are used in fluorescence-lifetime imaging microscopy. Labeling with organic fluorescent dyes requires purification, chemical modification, and intracellular injection of a host protein.

GFP variants can be attached to a host protein by genetic engineering which can be more convenient. To avoid this drawback, bioluminescence resonance energy transfer or BRET has been developed.

An introduction to the fluorescence study of the kinetics of energy

One drawback of BRET is the requirement to generate at least one fusion protein encoding luciferase, though some applications of FRET can be implemented with antibody-conjugated fluorophores. This luciferase is smaller 19 kD and brighter than the more commonly used luciferase from Renilla reniformis.

In many biological situations, however, researchers might need to examine the interactions between two, or more, proteins of the same type—or indeed the same protein with itself, for example if the protein folds or forms part of a polymer chain of proteins [34] or for other questions of quantification in biological cells.

Yet researchers can detect differences in the polarisation between the light which excites the fluorophores and the light which is emitted, in a technique called FRET anisotropy imaging; the level of quantified anisotropy difference in polarisation between the excitation and emission beams then becomes an indicative guide to how many FRET events have happened.

The applications of fluorescence resonance energy transfer FRET have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields.

FRET can be used as spectroscopic ruler in various areas such as structural elucidation of biological molecules and their interactions in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterial etc.

Based on the mechanism of FRET a variety of novel chemical sensors and biosensors have been developed. An alternative method to detecting protein—protein proximity is the bimolecular fluorescence complementation BiFCwhere two parts of a fluorescent protein are each fused to other proteins.

When these two parts meet, they form a fluorophore on a timescale of minutes or hours.The second area of this study, in Chapter III to Chapter V, presents the applications of these nanostructures in fundamental studies, i.e.

the mechanisms of plasmon enhanced fluorescence and photo-oxidation kinetics of CdSe quantum dots, and. Introduction to Fluorescence Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism.

Structure-Specific Intrinsic Fluorescence of Protein Amyloids Used to Study their Kinetics of Aggregation Fiona T.S. Chan, Dorothea Pinotsi, Gabriele S. Kaminski Schierle and Clemens F.

Kaminski University of Cambridge, Cambridge, United Kingdom Chapter 13 INTRODUCTION Fluorescence techniques are among the most powerful in the study of. Chlorophyll a fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II Xin-Guang Zhu1, Govindjee2, Neil Baker3, Eric de Sturler4, Donald R.

Ort5, Stephen P. Long1 1. Fluorescence resonance energy transfer (FRET) FRET utilizes non-radiative energy transfer from a donor fluorophore to an acceptor fluorophore resulting in fluorescence emission from the acceptor.

FRET can occur where there is sufficient overlap of the donor emission spectrum with the acceptor excitation spectrum, close proximity . APPLICATION NOTE Study of Fluorescence Quenching Kinetics Using Stopped-Flow resolution of ms which greatly improves the quality of the recorded kinetic and enables accurate fitting of the decay and.

Introduction to Fluorescence Microscopy | Protocol