Some features of this site are not supported in the browser you are using

Aspects of the video functionality may not work in Internet Explorer 7 and below. For an optimum user experience, please consider upgrading your browser.

Fluorescence Assays in Drug Discovery

Jack Owicki

Drug Discovery & Development

The course describes the principles of fluorescence assays and their application in fields related to drug discovery, where samples are small in volume, large in number, and presented in microplates.

Product description

Fluorescence is the detection method most often used for assays in drug discovery and related biomedical applications. The scientists and technicians who create and run the assays often do not have a firm grounding in the technique, which leads to wasted resources and opportunities.

This course aims to provide a grounding in the principles of fluorescence as they apply to assays, a discussion of the strengths and weaknesses of fluorescence assays, and a survey of the different types of fluorescence assays. Examples of assay targets include enzymes, ligand-receptor binding, DNA quantitation, and signal transduction in living cells.

The course is most commonly taken by biologists (B.S. to Ph.D. level), but it is also useful for physical scientists and engineers who in one way or another work with these assays and wish to understand them better.

You have to purchase this package to gain access to any associated resources

Premium content

Day One

Module 1: Introduction and Principles of Fluorescence

This module includes an overview of the course, including topic covered and a bit on where fluorescence relates to other types of optical detection strategies. It describes what happens at a molecular level and how you take the principal kinds of fluorescence measurements and spectra.

Module 2: Examples of Fluorophores That Are Relevant to Drug Discovery

This module is a survey of some of the different kinds of molecules that are used as fluorescent labels, from organic molecules like fluorescein to fluorescent proteins and quantum dots.

Module 3: Fluorescence Instruments

First, this module presents the different types of fluorometric instruments, not from a commercial standpoint but according to their basic architectures and analytical goals. Second, it discusses the behaviour of instrument components like light sources, filters, and detectors.

Module 4: Fluorescence Labelling Chemistries

A brief discussion of the main techniques for putting fluorescent labels on the amino and sulfhydryl groups of proteins, the effects of multiple labels on one protein, and the principal methods for getting fluorescent labels inside living cells without disrupting biological function.

Module 5: Interferences and Limitations on Fluorescence Assays

Fluorescence assays can be very powerful, but they do suffer from some common problems. This module discusses some of those difficulties and briefly reviews fluorometric methods based on fluorogenic substrates.

Module 6: Fluorometric Methods Based on Fluorogenic Substrates and on Image Analysis

Enzyme assays based on fluorogenic substrates are covered briefly, and then assays based on image analysis are discussed at some length. High-resolution images of cells that are labelled very specifically can reveal a lot of information about biological function that is hard to get by other means. This is often called High Content Screening or High Content Analysis. We’ll concentration on its use to probe signal transduction and receptor desensitization, while touching on some other related applications.

Module 7: Fluorometric Methods Based on Environmental Changes in Fluorescence Intensity

Some fluorophores are very sensitive to their environment. They may be dim in water but bright when bound to a generic protein or nucleic acid. They may change their intensity or spectral wavelengths depending on whether they’re bound to an ion like Ca++. We’ll discuss these topics, concentrating especially on the ion-sensitive fluorophores and on a method of detecting changes in membrane potential.

Module 8: Fluorometric Methods based on Foerster Resonance Energy Transfer (FRET) - Part One

FRET is a much-employed method in which an excited fluorophore may donate its energy (picked up by absorbing a photon) to a nearby chromophore, enabling many different types of proximity assays. We’ll talk about the molecular principles of FRET and about several different applications, including fluorogenic substrates, Taqman®, and membrane-potential sensing. Time-resolved applications of FRET are reserved until a later module.

Module 8: Fluorometric Methods based on Foerster Resonance Energy Transfer (FRET) - Part Two

FRET is a much-employed method in which an excited fluorophore may donate its energy (picked up by absorbing a photon) to a nearby chromophore, enabling many different types of proximity assays. We’ll talk about the molecular principles of FRET and about several different applications, including fluorogenic substrates, Taqman®, and membrane-potential sensing. Time-resolved applications of FRET are reserved until a later module.

Module 9: Fluorometric Methods Based on Fluorescence Lifetime

There’s a time interval, called the fluorescence lifetime, between the absorption of a photo and its emission as fluorescence. Since this interval varies among fluorophores (and even depending on the environment for a given fluorophore), lifetime can in various ways be made the basis of fluorescence assays.

Module 10: Fluorometric Methods Based on Time-Resolved Fluorescence and Time-Resolved FRET

We’ll concentration on Time Resolved Fluorescence, where long-lived lanthanide-ion labels are excited with pulses of light, and especially applications where time-resolved fluorescence is coupled with FRET. There are many applications, but we’ll concentrate on ligand-receptor binding assays.

Module 11: Fluorometric Methods Based on Fluorescence Polarization or Anisotropy

Fluorescence polarization (and its close relative, fluorescence anisotropy) enable assays that distinguish between small, rapidly rotating labelled molecules and larger, slowly rotating ones. We’ll talk about the principles of the method and its range of applicability. Examples will include ligand-receptor binding, protease, and protein kinase assays.

Module 12: Fluorometric Methods Based on Fluorescence Fluctuation Spectroscopies

As a very small number of labelled molecules diffuse through the ~1 femtoliter (1 um^3) volume of the focal point of a fluorescence confocal microscope, they cause fluctuating fluorescence intensities that can be analysed by a variety of sophisticated methods to extract information about molecular size and brightness; this in turn can be used to create powerful assays that work well in tiny volumes. We’ll have a brief survey of the ideas behind these assays and then a couple of examples of their application.