What is luminescence?
Luminescence is the emission of light by a substance as a result of a chemical reaction (chemiluminescence) or an enzymatic reaction (bioluminescence).
Luminescence detection is optically simpler than fluorescence detection as it does not require a light source or specific optics for excitation.
Figure 1 - Flash and Glow reaction
Luminescence can be either a ‘flash’ or a ‘glow’ reaction, depending on the kinetic profiles. Flash luminescence gives a very bright signal for a short amount of time, usually seconds. Glow luminescence emits a more stable but usually less intense signal that can last for several minutes or hours. Flash luminescence generally requires a detection system with injectors that can deliver substrate to the reaction shortly before taking a measurement so that signal is not missed. White microplates are usually recommended for luminescence as they reflect light and maximize the signal. (see Figure 1)
Advantages of luminescence
Luminescence is an extremely popular detection platform for many applications compared to absorbance and fluorescence. It generally offers a wider dynamic range and a higher sensitivity as background interference (autofluorescence from compounds, media and cells) is low. In addition, luminescence assays often use a homogenous (no wash) protocol, which makes them simpler to automate for high-throughput applications.
Luminescence Overview
Luminescence definitions
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How does luminescence detection work?
Unlike fluorescence, where a molecule emits light upon excitation by an external light source, luminescence is generated by a chemical or biological reaction, for example an enzyme and its substrate. The resulting light can be detected by a photomultiplier tube (PMT), in which photons are converted to electrons, with the resulting current proportional to the amount of light. Measurement of the signal is expressed as relative light units (RLU).
How does a luminometer work?
When a luminescence reaction is set up in a microplate, a luminometer (or luminescence microplate reader), is used to measure the amount of light produced. The microplate is placed in a light-tight read chamber, and light from each well is detected in turn by a PMT. The luminescence readings are expressed as RLU.
In most luminescence applications the total light produced by the sample is measured, without the selection of particular wavelengths. But for other applications like BRET, a luminescence plate reader can be equipped with filters or monochromators that allow the selection of specific wavelengths in order to measure binding or other biomolecular events.
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NanoBRET/BRET
BRET (bioluminescence resonance energy transfer) is a technique for measuring protein-protein or protein-ligand interactions that involves the interaction of a bioluminescent donor and a fluorescent acceptor.
Detection of NanoBRET signals, and analysis of the resulting data, requires sensitive instrumentation and advanced software. Register to learn more:
Single/Dual -Luciferase Reporter Gene Assays
Reporter gene assays are important tools for studying gene expression associated with the activation of cellular pathways. Cells are transfected with a plasmid containing the reporter gene and a sequence of interest, typically a promoter or other transcriptional control element. When the promoter is activated, the reporter gene is expressed and its levels can be measured.
Learn more about dual luciferase reporter gene assay:
- Monitor NF-kB activation with a sensitive dual reporter assay on the SpectraMax iD5 microplate reader*
- Detect dual luciferase expression on the FlexStation 3 microplate reader
- Dual-Luciferase assays in 96- and 384-well formats on the SpectraMax L
- Measuring luciferase expression using the SpectraMax Glo Steady-Luc Reporter Assay Kit
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Customer Breakthrough
University of Geneva uses the SpectraMax® L Microplate Reader for luciferase-based assays
To support vital work at the University of Geneva, M.Yves Cambet’s group use a variety of state-of-the-art microplate readers including the SpectraMax L for luciferase-based assays.
Reactive Oxygen Species (ROS)
Reactive Oxygen Species (ROS) are chemically reactive molecules containing oxygen. In eukaryotes, these molecules are mainly created during aerobic respiration and can cause problems, such as DNA damage and lipid peroxidation, resulting in cellular damage.
We demonstrate how SpectraMax microplate readers can be used to accurately quantify ROS levels using a luminescence-based assay:
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Cytotoxicity Assays
Cytotoxicity is often measured in response to an experimental treatment or potential drug. Having a way to easily screen cytotoxicity in treated cells is critical to identifying new therapeutic treatments or understanding cellular signaling pathways that affect cell health. Common indicators of cytotoxicity include the ATP level in a cell population and integrity of cellular membranes, both of which can be measured using a variety of microplate-based assays.
Learn more about cytotoxicity in cells:
ATP-based cell viability assays
Luminescent ATP assays for cell viability and cytotoxicity provide a sensitive and rapid way to measure the number of viable cells in culture and quantify the cytotoxic effects of experimental treatments. These assays can be done in a high-throughput format using SpectraMax® microplate readers.
Learn about the CellTiter-Glo Luminescent Cell Viability Assay and more:
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Chemiluminescent ELISA
Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides that have been implicated in mammalian vascular development and in disease processes involving abnormal blood vessel growth.
Learn more about chemiluminescent ELISA method using our luminometer and data acquisition and analysis software:
Mycoplasma Monitoring
Mycoplasma, the smallest and simplest of the prokaryotes, are common contaminants of cell cultures. Symptoms of mycoplasma contamination include a reduction in the rate of proliferation and changes in cellular responses, including gene expression
Learn how the MycoAlert Assay and MycoAlert PLUS Assay from Lonza provide a rapid and convenient way to detect viable mycoplasma in cell cultures using a luminescence microplate reader
Resources of Luminescence
Publications
The Best Microplate Readers According to our Product Reviewers
The Best Microplate Readers According to our Product Reviewers
According to Biocompare, “one of the most overlooked pieces of equipment in any lab is the microplate reader. While they play an important role in generating data from a wide range of a…
Application Note
Spectral Fusion™ Illumination technology for an extended dynamic range on the SpectraMax i3x Multi-Mode Microplate Reader
Spectral Fusion™ Illumination technology for an extended dynamic range on the SpectraMax i3x Multi-Mode Microplate Reader
Many microplate readers and spectrophotometers use a photomultiplier tube (PMT) to detect fluorescence and luminescence signals. PMTs work by initially detecting photons from the…
Application Note
Rapidly assess drug response in 2D and 3D breast cancer model systems with a luminescent viability assay
Rapidly assess drug response in 2D and 3D breast cancer model systems with a luminescent viability assay
For many years, two-dimensional (2D) cell culture involving cells grown in a monolayer on a flat culture surface has served as a convenient system for investigating disease mechanisms…
eBook
Virology and Vaccine Research Microplate Reader Solutions
Virology and Vaccine Research Microplate Reader Solutions
We understand what a challenging time this is for those who are working to get accelerated testing and medicines to the market as soon as possible and are committed to supporting scient…
eBook
The Ultimate Guide to Microplate Reader Solutions
The Ultimate Guide to Microplate Reader Solutions
Evaluating microplate readers doesn’t have to be overwhelming. First, consider your application needs. If your budget is modest, a single-mode reader dedicated to your main application…
Blog
FDA 21 CFR Part 11 and the importance of regulatory compliance in GMP and GLP labs
FDA 21 CFR Part 11 and the importance of regulatory compliance in GMP and GLP labs
The regulations for food and drug in the United States, described in the Title 21 of the Code of Federal Regulations, and the EudraLex Annex 11 in EU, are critical in ensuring safe…
Blog
Celebrating International Women’s Day
Celebrating International Women’s Day
Monday, March 8, 2021 is International Women’s Day (IWD). The theme of this year’s IWD is #ChooseToChallenge--encouraging us to call out gender bias and inequality and to seek out…
Publications
Multiple Steps from Microplate-Reader Workstations
Multiple Steps from Microplate-Reader Workstations
Scientists take many roads to overcoming bottlenecks in workflows. For example, a workstation including a microplate reader can handle multiple steps, which improves efficiency and adds…
Application Note
Measure p53-MDM2 protein interaction with NanoBRET technology
Measure p53-MDM2 protein interaction with NanoBRET technology
We describe validation of the SpectraMax® iD5 reader with the NanoBRET™ PPI Control Pair, consisting of the interacting protein partners p53 and MDM2.
Application Note
Monitor NF-κB activation with a sensitive dual luciferase reporter assay on the SpectraMax iD5
Monitor NF-κB activation with a sensitive dual luciferase reporter assay on the SpectraMax iD5
Reporter genes are valuable tools for studying gene expression, serving as surrogates for genes involved in various signaling pathways and disease conditions. Luciferases are the most…
Application Note
Detect dual luciferase expression on the FlexStation 3 microplate reader
Detect dual luciferase expression on the FlexStation 3 microplate reader
Reporter gene assays are important tools for studying gene expression associated with the activation of cellular pathways. Cells are transfected with a plasmid containing the reporter…
Application Note
Measuring reactive oxygen species with SpectraMax microplate readers
Measuring reactive oxygen species with SpectraMax microplate readers
Reactive Oxygen Species (ROS) are chemically reactive molecules containing oxygen. In eukaryotes, these molecules are mainly created during aerobic respiration and can cause problems,…
Application Note
MycoAlert Mycoplasma Detection Assays on Molecular Devices Microplate Readers
MycoAlert Mycoplasma Detection Assays on Molecular Devices Microplate Readers
Mycoplasma, the smallest and simplest of the prokaryotes, are common contaminants of cell cultures. Symptoms of mycoplasma contamination include a reduction in the rate of…
Application Note
Cytotoxicity in Cells: Easy Determination Using Lonza ViaLight Plus and ToxiLight BioAssays on the SpectraMax L Microplate Luminometer
Cytotoxicity in Cells: Easy Determination Using Lonza ViaLight Plus and ToxiLight BioAssays on the SpectraMax L Microplate Luminometer
Bioluminescent cytotoxicity assays offer the user increased detection limits, speed, and accuracy, and have been well documented. Healthy cells maintain a high constant ATP…
Application Note
Dual-Luciferase assays in 96- and 384-well formats on the new SpectraMax L
Dual-Luciferase assays in 96- and 384-well formats on the new SpectraMax L
Reporter gene assays are used to study the expression of eukaryotic genes. In dual reporter gene assays cells are transfected with two vectors, the first containing an experimental…
Application Note
Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer
Chemiluminescent VEGF ELISA Using the SpectraMax L Microplate Luminometer
Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides that have been implicated in mammalian vascular development and in disease processes involving…
Application Note
Luminescent cell viability and cytotoxicity assays on the SpectraMax i3x Multi-Mode Microplate Reader
Luminescent cell viability and cytotoxicity assays on the SpectraMax i3x Multi-Mode Microplate Reader
The CellTiter-Glo assay from Promega uses luciferase enzyme, which requires ATP in order to generate light. The luminescent signal produced in the assay depends on the amount of ATP in…
Application Note
NanoBRET Technology on the SpectraMax i3x Multi-Mode Microplate Reader
NanoBRET Technology on the SpectraMax i3x Multi-Mode Microplate Reader
Protein-protein interactions (PPI) are vital to cellular function and signaling. Many methods have been employed to study PPI, including co-immunoprecipitation and pull-down assays, as…
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