Plate reader

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Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, [1] bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 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.

Contents

Methods

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 [2] (i.e. in the MTT assay for cell viability). [3] 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. Several conventional colorimetric analyses have been miniaturized to function quantitatively in a plate reader, with performance suitable for research purposes. Examples of analyses converted to plate reader methods include several for ammonium, nitrate, nitrite, [4] urea, [5] iron(II), [6] and orthophosphate. [7] More recent colorimetric chemistries have been developed directly for use in plate readers. [8]

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. [9]

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 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.[ citation needed ]

Common applications include luciferase -based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of ATP. [10]

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.[ citation needed ]

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.[ citation needed ]

Light scattering and nephelometry

Light scattering and nephelometry are methods for the determination of the cloudiness of a solution (i.e.: insoluble particles in a solution). A light beam passes through the sample and the light is scattered by the suspended particles. The measured forward scattered light indicates the amount of the insoluble particles present in solution. Common nephelometry/light scattering applications include automated HTS drug solubility screening, long-term microbial growth kinetics, flocculation, aggregation and the monitoring of polymerization and precipitation, including immunoprecipitation.[ citation needed ]

Instruments and assays

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). There are also instruments for measuring the dynamic or static light scattered from samples in a microplate. 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:

Related Research Articles

<span class="mw-page-title-main">Fluorescence</span> Emission of light by a substance that has absorbed light

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation. A perceptible example of fluorescence occurs when the absorbed radiation is in the ultraviolet region of the electromagnetic spectrum, while the emitted light is in the visible region; this gives the fluorescent substance a distinct color that can only be seen when the substance has been exposed to UV light. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after.

<span class="mw-page-title-main">Raman spectroscopy</span> Spectroscopic technique

Raman spectroscopy is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.

<span class="mw-page-title-main">Ultraviolet–visible spectroscopy</span> Range of spectroscopic analysis

UV spectroscopy or UV–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. Being relatively inexpensive and easily implemented, this methodology is widely used in diverse applied and fundamental applications. The only requirement is that the sample absorb in the UV-Vis region, i.e. be a chromophore. Absorption spectroscopy is complementary to fluorescence spectroscopy. Parameters of interest, besides the wavelength of measurement, are absorbance (A) or transmittance (%T) or reflectance (%R), and its change with time.

<span class="mw-page-title-main">X-ray fluorescence</span> Emission of secondary X-rays from a material excited by high-energy X-rays

X-ray fluorescence (XRF) is the emission of characteristic "secondary" X-rays from a material that has been excited by being bombarded with high-energy X-rays or gamma rays. The phenomenon is widely used for elemental analysis and chemical analysis, particularly in the investigation of metals, glass, ceramics and building materials, and for research in geochemistry, forensic science, archaeology and art objects such as paintings.

<span class="mw-page-title-main">Spectrophotometry</span> Branch of spectroscopy

Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. Spectrophotometry uses photometers, known as spectrophotometers, that can measure the intensity of a light beam at different wavelengths. Although spectrophotometry is most commonly applied to ultraviolet, visible, and infrared radiation, modern spectrophotometers can interrogate wide swaths of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, and/or microwave wavelengths.

<span class="mw-page-title-main">Fluorescence spectroscopy</span> Type of electromagnetic spectroscopy

Fluorescence spectroscopy is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores.

<span class="mw-page-title-main">Photometer</span> Instrument to measure light intensity

A photometer is an instrument that measures the strength of electromagnetic radiation in the range from ultraviolet to infrared and including the visible spectrum. Most photometers convert light into an electric current using a photoresistor, photodiode, or photomultiplier.

Laser-induced fluorescence (LIF) or laser-stimulated fluorescence (LSF) is a spectroscopic method in which an atom or molecule is excited to a higher energy level by the absorption of laser light followed by spontaneous emission of light. It was first reported by Zare and coworkers in 1968.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

A spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, and the emission is observed either at a single wavelength, or a scan is performed to record the intensity versus wavelength, also called an emission spectrum. The instrument is used in fluorescence spectroscopy.

Ultrafast laser spectroscopy is a spectroscopic technique that uses ultrashort pulse lasers for the study of dynamics on extremely short time scales. Different methods are used to examine the dynamics of charge carriers, atoms, and molecules. Many different procedures have been developed spanning different time scales and photon energy ranges; some common methods are listed below.

Fluorescence anisotropy or fluorescence polarization is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. Early pioneers in the field include Aleksander Jablonski, Gregorio Weber, and Andreas Albrecht. The principles of fluorescence polarization and some applications of the method are presented in Lakowicz's book.

<span class="mw-page-title-main">Fluorometer</span>

A fluorometer, fluorimeter or fluormeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium. Modern fluorometers are capable of detecting fluorescent molecule concentrations as low as 1 part per trillion.

<span class="mw-page-title-main">Fluorescence in the life sciences</span> Scientific investigative technique

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers. TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET. The resulting assay provides an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false positive/false negative results. FRET involves two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength.

A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.

<span class="mw-page-title-main">Fluorescence polarization immunoassay</span> Class of invitro biochemical test

Fluorescence polarization immunoassay (FPIA) is a class of in vitro biochemical test used for rapid detection of antibody or antigen in sample. FPIA is a competitive homogenous assay, that consists of a simple prepare and read method, without the requirement of separation or washing steps.

Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.

<span class="mw-page-title-main">Fluorescence imaging</span> Type of non-invasive imaging technique

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

References

  1. ↑ Neves, Bruno Junior; Agnes, Jonathan Paulo; Gomes, Marcelo do Nascimento; Henriques Donza, Marcio Roberto; Gonçalves, Rosângela Mayer; Delgobo, Marina; Ribeiro de Souza Neto, Lauro; Senger, Mario Roberto; Silva-Junior, Floriano Paes; Ferreira, Sabrina Baptista; Zanotto-Filho, Alfeu; Andrade, Carolina Horta (March 2020). "Efficient identification of novel anti-glioma lead compounds by machine learning models". European Journal of Medicinal Chemistry. 189: 111981. doi:10.1016/j.ejmech.2019.111981. PMID   31978780. S2CID   210892159.
  2. ↑ Ashour, Mohamed-Bassem A.; Gee, Shirley J.; Hammock, Bruce D. (November 1987). "Use of a 96-well microplate reader for measuring routine enzyme activities". Analytical Biochemistry. 166 (2): 353–360. doi:10.1016/0003-2697(87)90585-9. PMID   3434778.
  3. ↑ Mosmann, Tim (December 1983). "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays". Journal of Immunological Methods. 65 (1–2): 55–63. doi:10.1016/0022-1759(83)90303-4. PMID   6606682.
  4. ↑ Sims, G. K.; Ellsworth, T. R.; Mulvaney, R. L. (11 November 2008). "Microscale determination of inorganic nitrogen in water and soil extracts". Communications in Soil Science and Plant Analysis. 26 (1–2): 303–316. doi:10.1080/00103629509369298.
  5. ↑ Greenan, N.S.; Mulvaney, R.L.; Sims, G. K. (11 November 2008). "A microscale method for colorimetric determination of urea in soil extracts". Communications in Soil Science and Plant Analysis. 26 (15–16): 2519–2529. doi:10.1080/00103629509369465.
  6. ↑ Tor, Jason M.; Xu, Caifen; Stucki, Joseph M.; Wander, Michelle M.; Sims, Gerald K. (August 2000). "Trifluralin Degradation under Microbiologically Induced Nitrate and Fe(III) Reducing Conditions". Environmental Science & Technology. 34 (15): 3148–3152. Bibcode:2000EnST...34.3148T. doi:10.1021/es9912473.
  7. ↑ D'Angelo, Elisa; Crutchfield, J.; Vandiviere, M. (November 2001). "Rapid, Sensitive, Microscale Determination of Phosphate in Water and Soil". Journal of Environmental Quality. 30 (6): 2206–2209. doi:10.2134/jeq2001.2206. PMID   11790034.
  8. ↑ Rhine, E. D.; Mulvaney, R. L.; Pratt, E. J.; Sims, G. K. (1998). "Improving the Berthelot Reaction for Determining Ammonium in Soil Extracts and Water". Soil Science Society of America Journal. 62 (2): 473. Bibcode:1998SSASJ..62..473R. doi:10.2136/sssaj1998.03615995006200020026x.
  9. ↑ Lin, Kedan; Sadée, Wolfgang; Mark Quillan, J. (February 1999). "Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader". BioTechniques. 26 (2): 318–326. doi: 10.2144/99262rr02 . PMID   10023544.
  10. ↑ Lin, Kedan; Sadée, Wolfgang; Mark Quillan, J. (February 1999). "Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader". BioTechniques. 26 (2): 318–326. doi: 10.2144/99262rr02 . PMID   10023544.
  11. ↑ Ashour, Mohamed-Bassem A.; Gee, Shirley J.; Hammock, Bruce D. (November 1987). "Use of a 96-well microplate reader for measuring routine enzyme activities". Analytical Biochemistry. 166 (2): 353–360. doi:10.1016/0003-2697(87)90585-9. PMID   3434778.
  12. ↑ "AlphaScreen | BMG LABTECH".
  13. ↑ Suprun, Maria; Getts, Robert; Raghunathan, Rohit; Grishina, Galina; Witmer, Marc; Gimenez, Gustavo; Sampson, Hugh A.; Suárez-Fariñas, Mayte (5 December 2019). "Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy". Scientific Reports. 9 (1): 18425. Bibcode:2019NatSR...918425S. doi:10.1038/s41598-019-54868-7. PMC   6895130 . PMID   31804555.
  14. ↑ Shin, Hye Ji; Kwak, Minjeong; Joo, Sihwa; Lee, Ji Youn (2022). "Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader". Scientific Reports. 12 (1): 20146. Bibcode:2022NatSR..1220146S. doi:10.1038/s41598-022-24480-3. PMC   9684140 . PMID   36418509.
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