Fluorescence & Luminescence Assays

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Fluorescence and luminescence based readouts are keystones in virtually all biological assay development and screening. At Charnwood Discovery we have extensive experience in the use and application of both for developing bespoke drug discovery assays. 

Fluorescence

Fluorescence is a form of photo-luminescence in which light is emitted from an excited fluorophore at a longer wavelength (and therefore energy) than the light used to excite it. 

The excitation-emission process is near-instantaneous (nanoseconds) and is therefore very short lived – a consideration when it comes to induction and detection.

Fluorescence

At Charnwood Discovery we use fluorescence in a wide range of applications – from simple plate-based biochemical assays to complex live cell imaging experiments.

A key benefit of fluorescence is the flexibility and versatility afforded by the high number of possible fluorophores and wavelengths of light. Using non-overlapping wavelengths of light allows detection of multiple different readouts from single samples through multiplexing.

Something we need to consider and account for with fluorescence-based assays is possible background interference from either the system being worked with (cells, media, buffers) or sometimes even the inherent fluorescence of compounds themselves. This can reduce assay windows and result in false negative/positive results.

 

Fluorescence luminescence

Luminescence

By contrast, ‘luminescence’ is a form of chemi-luminescence in which the detected light emission is induced instead by a chemical reaction. This signal generation is therefore as long lived as the chemical induction reaction and the components themselves – an important consideration with regards to assay development and conditions. 

Fluorescence luminescence

Luminescence has an equally wide variety of applications – common ones in our hands include reporter assays in which we measure changes in gene expression, detecting changes to protein expression through immunoblotting and measuring cell viability by monitoring ATP levels using CellTiter-Glo®. 

A key advantage of luminescence-based assays is low levels of background interference from the assay system or compounds themselves. As a result, luminescence assays can demonstrate very high levels of assay performance and sensitivity. 

With luminescence assays it is always important to consider the possible consumption and exhaustion of the detection reagent itself.

Fluorescence vs. Luminescence

There are advantages and disadvantages to the use of fluorescence vs. luminescence readouts in biological assay development and screening. At Charnwood Discovery we are experienced in applying one or both at the appropriate times within a project.

See examples of our work with both types of assay on the listed pages below:

  • Cell viability assays (Cell titer-Glo and RealTime-Glo)
  • FRET
  • HTRF
  • NanoBRET
  • Luminescence-based cell reporter assays
  • Cyp P450 Glo