What is fluorescence?
Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength (the excitation: Ex) followed by a short-lived emission (Em) of light at a longer wavelength (Figure 2). The distance between the excitation and emission peaks is known as the Stokes shift and is fluorophore dependent (Figure 1).
Fluorescence involves an external light source to excite the sample at a particular wavelength. When excited at the appropriate wavelength, the molecule is transformed from a ground to an excited state. As the molecule returns to the ground state, energy is released in the form of heat (loss of energy) and light at a different longer wavelength of lower energy (Figure 3).
Figure 1
Figure 2
Figure 3
How does fluorescence detection work?
A microplate reader with fluorescence intensity (FI) detection uses a light source, usually a Xenon flash lamp or LED, to excite a fluorophore (fluorescent molecule) at a particular wavelength. The wavelength required to excite the sample can be selected using either a filter of a specific wavelength or a monochromator tuned to the required wavelength.
The fluorophore then emits light of a different wavelength, selected by a second filter or monochromator. This emitted fluorescence is detected by a photomultiplier tube (PMT), and the fluorescence intensity of the sample is expressed as relative fluorescence units.
Fluorescence Overview
Fluorescence definitions
Customer Citations
Our SpectraMax Gemini Microplate Reader has more than 10,400 and counting
Fluorescence applications
- Calcium Assays (GPCR)
- cAMP Assays (GPCR)
- Caspase-3 Apoptosis Assays
- Cell Proliferation Assays
- Cytotoxicity Assays
- EarlyTox Cardiotoxicity Assay
- EarlyTox Cell Viability Assay Kits
- Fluorescent Protein Quantitation
- Nanoparticle Analysis
- Neuronal Calcium Flux
- Nucleic Acid Quantitation
- Tryptophan Detection
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Fluorometer vs spectrofluorometer vs fluorescence plate reader
There are many terms that refer to instruments used to measure the fluorescence of a sample: fluorometer, spectrofluorometer, fluorescence spectrophotometer, fluorescence spectrometer, fluorimeter, etc.
In general, they use a light source to excite the sample at a certain wavelength and measure the emitted fluorescence at a second wavelength. The sample may be held in many formats, including cuvettes, capillaries and petri dishes. ‘Fluorescence microplate reader’ refers to an instrument that can measure the fluorescence of samples in a microplate, most often a 96- or 384-well plate. Microplates offer higher throughput and are useful for screening or other applications where many samples must be analyzed.
What is a dual monochromator spectrofluorometer system?
Some fluorescence microplate readers use dual monochromators instead of filters to select the wavelength of light that excites the sample, as well as the wavelength that is emitted by the sample. Monochromators use a diffraction grating to spatially separate the colors of light and can select a wide range of wavelengths without the need to install a separate filter for each wavelength required by an application.
Our SpectraMax Gemini dual-monochromator spectrofluorometer uses two scanning monochromators for selection of the optimal excitation and emission wavelengths.
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Calcium Assays (GPCR)
Cell signaling via G protein-coupled receptors (GPCRs) can be assessed by monitoring the downstream effectors calcium or cyclic AMP (cAMP). Calcium flux in response to Gq protein-coupled receptor activation is commonly monitored in live cells in real time using calcium-sensitive dyes on a fluorescence plate reader.
Here are a few application notes on calcium assays (GPCR) you may find of interest:
cAMP Assays (GPCR)
Monitoring levels of cAMP, a second messenger produced in response to activation of adenylate cyclase, is one of the most common ways to screen for agonists and antagonists of Gi/Gs protein-coupled receptors. cAMP levels can be monitored using fluorescent molecules that bind to cAMP and are detected using a fluorescence plate reader.
Here are a few application notes on cAMP assays (GPCR) you may find of interest:
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Caspase-3 Apoptosis Assays
Apoptosis is a highly regulated cellular program that causes cell death in normal processes such as embryonic development, as well as diseases including cancer and neurodegenerative conditions. Assays for apoptosis can be performed using a variety of imaging or microplate reader detection systems and provide valuable information on normal and disease-related mechanisms of cell death.
Learn about a Caspase-3 single-step, homogenous assay that is specifically designed for microplate readers:
Cell Proliferation Assays
Quantitation of cell proliferation using fluorescence allows one to easily monitor the effects of drugs and other experimental treatments on cell growth.
This application note describes two assay methods of a cell prolfieration. In the first, cellular proliferation is quantitated using a cell-based standard curve. In the second, cellular proliferation is quantitated using RNase-treated cell samples and a DNA standard curve.
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Cytotoxicity Assays
Development of predictive in vitro assays suitable for safety and efficacy testing is of high interest for improving the drug development process and reducing drug attrition. There has been great interest in using stem cells as tools for screening compounds during early drug development.
In this scientific poster, we present results obtained from one of our multi-mode plate readers for performing toxicity assessment of various compounds using stem cell-derived cell models in a high throughput manner:
EarlyTox Cardiotoxicity Assay
Stem cell-derived human cardiomyocytes have phenotypic characteristics and electrophysiological profiles similar to those of native human cardiac cells. Cardiomyocytes in culture are able to form a beating syncytium, which behaves similarly to native cardiomyocytes. Oscillation of intracellular calcium levels occurring with synchronized contractions of the cells can be monitored using a calcium-sensitive dye, and treatment-induced changes in the pattern of oscillation can be monitored via changes in fluorescent signal over time.
Here is an application note you may find of interest:
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EarlyTox Cell Viability Assay Kits
Cell viability assays are critical to a broad spectrum of research areas ranging from investigation into the mechanisms of cell death to the development of new therapeutics targeting apoptosis in diseases. One of the most popular detection technologies for cell viability is a fluorescence microplate reader.
Here are a few applications you may find of interest:
- Measuring cell health on the SpectraMax iD3 reader with cell viability assays
- EarlyTox Live/Dead Assay Kit on SpectraMax Fluorescence Microplate Readers
- EarlyTox Caspase-3/7 R110 Assay Kit on SpectraMax Fluorescence Microplate Readers
- EarlyTox Glutathione Assay Kit on SpectraMax Fluorescence Microplate Readers
Fluorescent Protein Quantitation
Proteins inside eukaryotic cells exist in a dynamic state, in a highly-regulated balance between synthesis and degradation. Whereas protein synthesis is well-understood after decades of study, major advances in our knowledge of protein degradation have occurred only in the last two decades.
Learn more about fluorescent protein in our application note:
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Nanoparticle Analysis
Nanotechnology is a rapidly developing field that has caught the interest of the scientific community due to its potential applications in biomedical research.
Currently there are a limited number of techniques available to characterize nanoparticles and their molecular interactions. Here we describe spectral signature analysis as a method to confirm interactions between nanoparticles and surface coating molecules.
Neuronal Calcium Flux
Monitoring changes in intracellular calcium is an extremely useful technique for investigating the variety of roles that calcium ions play in functioning neurons. Direct measurement of the dynamic calcium flux within neuronal networks reveals how neurons process signals from the extracellular space.
This application note demonstrates the feasibility of measuring intracellular calcium flux in primary rat cortical neurons in a fluorescent microplate format:
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Nucleic Acid Quantitation
Quantitation of DNA is a critical step in molecular biology requiring accuracy, reliability, and the use of increasingly smaller sample volumes for applications such as next-generation sequencing. Compared to spectrophotometric DNA quantification, the fluorometric method provides key advantages such as significantly increased sensitivity, high selectivity for double-stranded DNA (dsDNA) over single-stranded DNA (ssDNA) or RNA, and improved contaminant tolerance (protein and carbohydrate molecules).
Here are a few applications notes on nucleic acid quantitation you may find of interest:
Tryptophan detection
The intrinsic fluorescence of proteins is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine. Tryptophan dominates the emission of proteins and is the most sensitive to solvent polarity and the local environment. Analysis of changes in tryptophan fluorescence can yield information on protein denaturation and conformation.
In these application notes, we demonstrate performance of the SpectraMax® multi-mode microplate readers for assays measuring intrinsic tryptophan fluorescence.
Resources of Fluorescence
Scientific Poster
Automated workflow for screening of CRISPR-edited cell lines. High throughput screening and analysis of monoclonality.
Automated workflow for screening of CRISPR-edited cell lines. High throughput screening and analysis of monoclonality.
Gene editing allows us to manipulate DNA through deletions, additions, or other modifications using several tools. There are numerous efficient strategies to carry out these modificatio…
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
Sensitive RNA fluorescent quantitation with the Quant-iT RiboGreen RNA assay kit
Sensitive RNA fluorescent quantitation with the Quant-iT RiboGreen RNA assay kit
Accurate quantification of nucleic acid concentration is important for downstream applications including transfection, cloning, PCR, and next generation sequencing (NGS). Often, these…
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…
Scientific Poster
Evaluation of a fluorescent approach to implement monoclonality assurance using calcein-AM viability dye
Evaluation of a fluorescent approach to implement monoclonality assurance using calcein-AM viability dye
Assessing monoclonality is key to the establishment of a cell line, and evidence of monoclonality is required by regulatory agencies to get a biopharmaceutical drug to the marketplace.
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
How to Implement Colony Picking Workflows with Automation
How to Implement Colony Picking Workflows with Automation
Colony picking represents a powerful tool within the workflow of mammalian cell line development and the study of microbiology.
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…
Application Note
Sensitive fluorescent quantitation of DNA with the Quant-iT PicoGreen dsDNA Assay Kit
Sensitive fluorescent quantitation of DNA with the Quant-iT PicoGreen dsDNA Assay Kit
Double-stranded DNA is typically quantitated in microplate readers by measuring the absorbance of the DNA solution at 260 nm. However, this method is only able to measure down to about…
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…
Publications
Advanced Cell Screening and Imaging Reveals How the Cardiac System Responds in Drug Development
Advanced Cell Screening and Imaging Reveals How the Cardiac System Responds in Drug Development
In drug discovery, time and monetary investments increase significantly the further candidates advance through the preclinical phase of testing, analysis and evaluation. By excluding no…
Publications
From 3D high throughput to simple automated imaging: Cellular imaging within reach of every laboratory
From 3D high throughput to simple automated imaging: Cellular imaging within reach of every laboratory
OUR ABILITY to image cells has come a long way since the pioneering days of Galileo Galilei and Antonie van Leeuwenhoek. The multiple imaging techniques available today range from simpl…
Publications
Everything you need to know about the ImageXpress Pico Automated Cell Imaging System
Everything you need to know about the ImageXpress Pico Automated Cell Imaging System
Depending on your application and exactly what you are researching, there are multiple imaging techniques available, ranging from confocal microscopy to electron microscopy and more - e…
Application Note
Calcium flux assay for in vitro neurotoxicity studies and drug screening
Calcium flux assay for in vitro neurotoxicity studies and drug screening
Calcium imaging, or monitoring changes in intracellular calcium, is an extremely useful technique for investigating the variety of roles that calcium ions have in functioning neurons.…
Application Note
Measure intrinsic tryptophan fluorescence on the SpectraMax iD3 microplate reader
Measure intrinsic tryptophan fluorescence on the SpectraMax iD3 microplate reader
The intrinsic fluorescence of proteins is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine. Tryptophan, which excites maximally around 270-280 nm and has an…
Application Note
Measuring cell health on the SpectraMax iD3 reader with cell viability assays
Measuring cell health on the SpectraMax iD3 reader with cell viability assays
Measuring cell health from the multiple vantage points of viability and apoptotic pathways can provide insight into the impacts of a variety of experimental treatments including drug…
Application Note
Intrinsic tryptophan detection with the SpectraMax i3 Multi-Mode Microplate Platform
Intrinsic tryptophan detection with the SpectraMax i3 Multi-Mode Microplate Platform
The intrinsic fluorescence of proteins is due to the aromatic amino acids tryptophan, tyrosine, and phenylalanine. Tryptophan, which excites maximally around 270-280 nm and has an…
Application Note
Complete cAMP workflow solution using the CatchPoint cAMP Fluorescent Assay Kit
Complete cAMP workflow solution using the CatchPoint cAMP Fluorescent Assay Kit
G-Protein Coupled Receptors (GPCRs) are important transmembrane proteins that translate extracellular signals into intracellular responses. These intracellular responses are comprised…
Application Note
Using the NanoOrange Protein Kit with SpectraMax Microplate Readers
Using the NanoOrange Protein Kit with SpectraMax Microplate Readers
This application note describes how to use the NanoOrange® Protein Quantitation Kit from Life Technologies in SpectraMax® microplate readers with the fluorescence detection mode and…
Application Note
Multiparametric assays for cardiomyocytes on a single platform
Multiparametric assays for cardiomyocytes on a single platform
Recently, it has been shown that changes in intracellular calcium flux associated with cardiomyocyte contractions can be monitored on a fluorescence imaging plate reader (FLIPR Tetra…
Application Note
EarlyTox Glutathione Assay Kit on SpectraMax Fluorescence Microplate Readers
EarlyTox Glutathione Assay Kit on SpectraMax Fluorescence Microplate Readers
Apoptosis is a highly regulated cellular program that causes cell death in normal processes such as embryonic development, as well as diseases including cancer and neurodegenerative…
Application Note
EarlyTox Live/Dead Assay Kit on SpectraMax Fluorescence Microplate Readers
EarlyTox Live/Dead Assay Kit on SpectraMax Fluorescence Microplate Readers
Cell viability assays are often performed to assess the effects of a variety of treatments, including drug candidates, pathway activators and inhibitors, and reporter genes. One of the…
Application Note
Optimization of a muscarinic M3-receptor assay using frozen CHO cells on the FlexStation 3 reader
Optimization of a muscarinic M3-receptor assay using frozen CHO cells on the FlexStation 3 reader
Cell-based assays can often be challenging and time consuming. To facilitate and streamline this complicated process, frozen cells, which can be assayed without prior cultivation, have…
Application Note
Calcium signaling with FLIPR Calcium 6 and 6-QF Assay Kits on the FlexStation 3 reader
Calcium signaling with FLIPR Calcium 6 and 6-QF Assay Kits on the FlexStation 3 reader
FLIPR® Calcium Assay Kits from Molecular Devices employ sensitive calcium indicators and proprietary masking dyes to enable researchers to conduct highly sensitive fluorescent screens…
Application Note
Quantitation of dsDNA samples using the SpectraMax Quant dsDNA Assay Kits
Quantitation of dsDNA samples using the SpectraMax Quant dsDNA Assay Kits
As different types of test samples may contain different concentrations of DNA, selection of an appropriate fluorescent reagent with the required sensitivity and detection range is…
Application Note
Measuring cell proliferation using the CyQUANT Cell Proliferation Assay with SpectraMax Microplate Readers
Measuring cell proliferation using the CyQUANT Cell Proliferation Assay with SpectraMax Microplate Readers
Quantitation of cell proliferation using fluorescence allows one to easily monitor the effects of drugs and other experimental treatments on cell growth. The CyQUANT Cell Proliferation…
Application Note
HTRF cAMP HiRange Assay on SpectraMax Multi-Mode Microplate Readers
HTRF cAMP HiRange Assay on SpectraMax Multi-Mode Microplate Readers
HTRF is a versatile technology developed by Cisbio Bioassays for detecting biomolecular interactions1. It combines fluorescence resonance energy transfer (FRET) technology with time-…
Application Note
Spectral signature analysis of surface functionalized nanoparticles
Spectral signature analysis of surface functionalized nanoparticles
Nanotechnology is a rapidly developing field that has caught the interest of the scientific community due to its potential applications in biomedical research. Currently there are a…
Application Note
Monitoring Gq protein-coupled receptor activation on the SpectraMax i3x reader with injector module
Monitoring Gq protein-coupled receptor activation on the SpectraMax i3x reader with injector module
Gq protein-coupled receptor activation is commonly monitored in live cells in real time using calcium-sensitive dyes on a fluorescence plate reader. Automated liquid handling within…
Application Note
EarlyTox Caspase-3/7 R110 Assay Kit on SpectraMax Fluorescence Microplate Readers
EarlyTox Caspase-3/7 R110 Assay Kit on SpectraMax Fluorescence Microplate Readers
Apoptosis is a highly regulated cellular program that causes cell death in normal processes such as embryonic development, as well as diseases including cancer and neurodegenerative…
Scientific Poster
High Throughput Cell Based Assays for Toxicity Assessment using SpectraMax® Paradigm Plate Reader
High Throughput Cell Based Assays for Toxicity Assessment using SpectraMax® Paradigm Plate Reader
Application note presents a comparison of assay performance between SpectraMax Paradigm reader read types, and the ImageXpress Micro imaging system.
Scientific Poster
Measurement of Proteosome Inhibition in Live Cells on the Analyst® GT, FLEXstation® and Gemini EM Microplate Readers Using the BD Biosciences Proteasome Sensor
Measurement of Proteosome Inhibition in Live Cells on the Analyst® GT, FLEXstation® and Gemini EM Microplate Readers Using the BD Biosciences Proteasome Sensor
The proteasome is a massive protein complex inside all eukaryotic cells (and some bacteria) that removes unnecessary proteins by breaking them down into short peptides. It consists of a…
Publications
Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells
Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells
A large percentage of drugs fail in clinical studies due to cardiac toxicity; thus, development of sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocyte…
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Monoclonality Assurance using Calcein-AM Viability Dye
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