Plate reader: Difference between revisions

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'''Plate readers''', also known as '''microplate readers''' or '''microplate photometers''', are instruments which are used to detect [[biology|biological]], [[chemistry|chemical]] or [[physics|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 61-1536 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 spectroscopy#Time-resolved fluorescence spectroscopy|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 [[ELISA]] assays, protein and nucleic acid quantification or enzyme activity assays<ref name="Mohamed-Bassem">{{cite journal|last=Mohamed-Bassem|first=A. Ashour|date=12 February 1987|title=Use of a 96-well microplatereadermicroplate reader for measuring routine enzyme activities|journal=[[Analytical Biochemistry]]|volume= 166|issue=2|pages=353–360|url=http://www.sciencedirect.com/science/article/pii/0003269787905859|doi=10.1016/0003-2697(87)90585-9}}</ref> (i.e. in the [[MTT assay]] for cell viability).<ref name="pmid6606682">

{{cite journal

| author = Mosmann, Tim

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==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 the 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 [[photomultiplier]] 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 the fluorescence intensity of [[calcium-sensitive dyes]] to assess intracellular calcium levels.

==Luminescence==

Luminescence is the result of a chemical or biochemical reaction. Luminescence detection is simpler optically than fluorescence detection because luminescence does not require a light source for excitation or optics for selecting discrete excitation wavelengths. A typical luminescence optical system consists of a light-tight reading chamber and a [[Photomultiplier|PMT]] detector. Some plate readers use an Analog PMT detector while others have a [[photon counting]] PMT detector. Photon Counting is widely accepted as the most sensitive means of detecting luminescence. Some plate readers offer filter wheel or tunable wavelength monochromator optical systems for selecting specific luminescent wavelengths. The ability to select multiple wavelengths, or even wavelength ranges, allows for detection of assays that contain multiple luminescent reporter enzymes, the development of new luminescence assays, as well as a means to optimize the signal to noise ratio.

Common applications include [[luciferase]] -based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of [[Adenosine triphosphate|ATP]].

==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-[[Förster resonance energy transfer|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==