Plate reader
Encyclopedia
Microplate Readers are laboratory instruments designed to detect biological
, chemical
or physical
events of samples in microtiter plate
s. They are widely used in research, drug discovery
, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 µL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence
intensity, luminescence
, time-resolved fluorescence, and fluorescence polarization.
assays, protein and nucleic acid quantification or enzyme activity assays (i.e. in the MTT assay
for cell viability). A light source illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), and a light detector located on the other side of the well measures how much of the initial (100 %) light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest.
tube (PMT). The advantages of fluorescence detection over absorbance detection are sensitivity, as well as application range, given the wide selection of fluorescent labels available today. For example, a technique known as calcium imaging
measures fluorescence intensity of calcium-sensitive dyes to asses intracellular calcium levels.
-based gene expression assays, as well as cell viability and cytotoxicity assays based on the luminescent detection of ATP
.
include Wyatt Technology and Malvern Technology. Another company, Harbinger Biotechnology and Engineering, specializes in an instrument for static light scattering.
Many of the detection modes (absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization) are available stand-alone in dedicated plate readers, but are very often found today combined into one instrument (multi-mode plate reader). The range of applications for multi-mode plate readers is extremely large. Some of the most common assays are:
While "plate reader" usually refers to the devices described above, many variations are available. Some examples of other devices working with the microplate format are:
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...
, chemical
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....
or physical
Physics
Physics is a natural science that involves the study of matter and its motion through spacetime, along with related concepts such as energy and force. More broadly, it is the general analysis of nature, conducted in order to understand how the universe behaves.Physics is one of the oldest academic...
events of samples in microtiter plate
Microtiter plate
A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
s. They are widely used in research, drug discovery
Drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 µL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
intensity, luminescence
Luminescence
Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold body radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal. This distinguishes luminescence from incandescence, which is light emitted by a...
, time-resolved fluorescence, and fluorescence polarization.
Absorbance
Absorbance detection has been available in microplate readers for more than 3 decades, and is used for assays such as ELISAELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
assays, protein and nucleic acid quantification or enzyme activity assays (i.e. in the MTT assay
MTT assay
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes to formazan dyes, giving a purple color. A main application allows to assess the viability and the proliferation of cells...
for cell viability). A light source illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), and a light detector located on the other side of the well measures how much of the initial (100 %) light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest.
Fluorescence
Fluorescence intensity detection has developed very broadly in the microplate format over the last two decades. The range of applications is much broader than when using absorbance detection, but instrumentation is usually more expensive. In this type of instrumentation, a first optical system (excitation system) illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator). As a result of the illumination, the sample emits light (it fluoresces) and a second optical system (emission system) collects the emitted light, separates it from the excitation light (using a filter or monochromator system), and measures the signal using a light detector such as a photomultiplierPhotomultiplier
Photomultiplier tubes , members of the class of vacuum tubes, and more specifically phototubes, are extremely sensitive detectors of light in the ultraviolet, visible, and near-infrared ranges of the electromagnetic spectrum...
tube (PMT). The advantages of fluorescence detection over absorbance detection are sensitivity, as well as application range, given the wide selection of fluorescent labels available today. For example, a technique known as calcium imaging
Calcium imaging
Calcium imaging is a scientific technique usually carried out in research which is designed to show the calcium status of a tissue or medium....
measures fluorescence intensity of calcium-sensitive dyes to asses intracellular calcium levels.
Luminescence
Luminescence detection is very popular for specific applications. The difference with fluorescence is that the light emitted by the samples is the result of a chemical or biochemical reaction (instead of being the result of excitation by light). Luminescence plate readers are simpler optically than fluorescence readers, as they don't require a light source, just a light detector. Typically, the optical system consists in a light-tight reading chamber, and PMT detector measuring the light emitted by the samples during the reaction. Common applications include luciferaseLuciferase
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P...
-based gene expression assays, as well as cell viability and cytotoxicity assays based on the luminescent detection of ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
.
Time-resolved fluorescence (TRF)
Time-resolved fluorescence (TRF) measurement is very similar to fluorescence intensity (FI) measurement. The only difference is the timing of the excitation / measurement process. When measuring FI, the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place. Even though emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that FI measurements always exhibit fairly elevated background signals. TRF offers a solution to this issue. It relies on the use of very specific fluorescent molecules, called lanthanides, that have the unusual property of emitting over long periods of time (measured in milliseconds) after excitation, when most standard fluorescent dyes (e.g. fluorescein) emit within a few nanoseconds of being excited. As a result, it is possible to excite lanthanides using a pulsed light source (Xenon flash lamp or pulsed laser for example), and measure after the excitation pulse. This results in lower measurement backgrounds than in standard FI assays. The drawbacks are that the instrumentation and reagents are typically more expensive, and that the applications have to be compatible with the use of these very specific lanthanide dyes. The main use of TRF is found in drug screening applications, under a form called TR-FRET (time-resolved fluorescence energy transfer). TR-FRET assays are very robust (limited sensitivity to several types of assay interference) and are easily miniaturized. Robustness, the ability to automate and miniaturize are features that are highly attractive in a screening laboratory.Fluorescence polarization
Fluorescence polarization measurement is also very close to FI detection. The difference is that the optical system includes polarizing filters on the light path: the samples in the microplate are excited using polarized light (instead of non-polarized light in FI and TRF modes). Depending on the mobility of the fluorescent molecules found in the wells, the light emitted will either be polarized or not. For example, large molecules (e.g. proteins) in solution, which rotate relatively slowly because of their size, will emit polarized light when excited with polarized light. On the other hand, the fast rotation of smaller molecules will result in a depolarization of the signal. The emission system of the plate reader uses polarizing filters to analyze the polarity of the emitted light. A low level of polarization indicates that small fluorescent molecules move freely in the sample. A high level of polarization indicates that fluorescent is attached to a larger molecular complex. As a result, one of the basic applications of FP detection is molecular binding assays, since they allow to detect if a small fluorescent molecule binds (or not) to a larger, non-fluorescent molecule: binding results in a slower rotation speed of the fluorescent molecule, and in an increase in the polarization of the signal.Light scattering
There are instruments for measuring the dynamic or static light scattered from samples in a microplate. Companies that sell plate readers for dynamic light scatteringDynamic light scattering
thumb|right|350px|Hypothetical Dynamic light scattering of two samples: Larger particles on the top and smaller particle on the bottomDynamic light scattering is a technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers...
include Wyatt Technology and Malvern Technology. Another company, Harbinger Biotechnology and Engineering, specializes in an instrument for static light scattering.
Many of the detection modes (absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization) are available stand-alone in dedicated plate readers, but are very often found today combined into one instrument (multi-mode plate reader). The range of applications for multi-mode plate readers is extremely large. Some of the most common assays are:
- ELISAELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
s - Protein and cell growth assays
- Nucleic acid quantitation
- Molecular interactions
- Enzyme activity
- Cell toxicity, proliferation, and viability
- ATP quantification
- Immunoassays
- High throughput screening of compounds and targets in drug discovery
While "plate reader" usually refers to the devices described above, many variations are available. Some examples of other devices working with the microplate format are:
- ELISPOTELISPOTThe Enzyme-linked immunosorbent spot assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983....
plate readers, used to count the colored spots that are formed in the course of ELISPOT assays. - High throughput imagers that can measure all the wells of a microplate at once
- High content screening (HCS) systems that image each well with high resolution, to look at cell populations
- Label-free instruments that use specialized microplates to measure binding events without the use of chemical markers