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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.
298:, 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-
171:, 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 ÎĽL 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 ÎĽL per well. Common detection modes for microplate assays are absorbance,
243:(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
130:
33:
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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
438:
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
320:
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
264:
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
242:
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
311:
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
293:
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
273:
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
329:
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
208:
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
321:
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.
302:
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.
247:
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
330:
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:
274:
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.
213:
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
610:
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".
401:
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:
2315:
978:
846:
Suprun, Maria; Getts, Robert; Raghunathan, Rohit; Grishina, Galina; Witmer, Marc; Gimenez, Gustavo; Sampson, Hugh A.; Suárez-Fariñas, Mayte (5 December 2019).
797:
Ashour, Mohamed-Bassem A.; Gee, Shirley J.; Hammock, Bruce D. (November 1987). "Use of a 96-well microplate reader for measuring routine enzyme activities".
482:
Ashour, Mohamed-Bassem A.; Gee, Shirley J.; Hammock, Bruce D. (November 1987). "Use of a 96-well microplate reader for measuring routine enzyme activities".
2207:
2186:
680:
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".
2296:
517:
Mosmann, Tim (December 1983). "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays".
552:
Sims, G. K.; Ellsworth, T. R.; Mulvaney, R. L. (11 November 2008). "Microscale determination of inorganic nitrogen in water and soil extracts".
1822:
645:
D'Angelo, Elisa; Crutchfield, J.; Vandiviere, M. (November 2001). "Rapid, Sensitive, Microscale
Determination of Phosphate in Water and Soil".
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971:
1883:
848:"Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy"
281:-based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of
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Label-free instruments that use specialized microplates to measure binding events without the use of chemical markers
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Absorbance detection has been available in microplate readers for more than 3 decades and is used for assays such as
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transmitted light will typically be related to the concentration of the molecule of interest. Several conventional
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Horta (March 2020). "Efficient identification of novel anti-glioma lead compounds by machine learning models".
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plate readers, used to count the colored spots that are formed in the course of ELISPOT assays.
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234:. More recent colorimetric chemistries have been developed directly for use in plate readers.
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assays, protein and nucleic acid quantification or enzyme activity assays (i.e. in the
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905:"Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader"
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of compounds and targets in drug discovery (Labeled Alpha Screen on most instruments)
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417:(HCS) systems that image each well with high resolution, to look at cell populations
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758:"Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader"
717:"Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader"
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High throughput imagers that can measure all the wells of a microplate at once
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581:"A microscale method for colorimetric determination of urea in soil extracts"
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detector. Some plate readers use an Analog PMT detector while others have a
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Shin, Hye Ji; Kwak, Minjeong; Joo, Sihwa; Lee, Ji Youn (2022).
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Lin, Kedan; Sadée, Wolfgang; Mark
Quillan, J. (February 1999).
715:
Lin, Kedan; Sadée, Wolfgang; Mark
Quillan, J. (February 1999).
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Greenan, N.S.; Mulvaney, R.L.; Sims, G. K. (11 November 2008).
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57:. Unsourced material may be challenged and removed.
585:Communications in Soil Science and Plant Analysis
554:Communications in Soil Science and Plant Analysis
265:consists of a light-tight reading chamber and a
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8:
151:, are instruments which are used to detect
1932:Nuclear magnetic resonance (NMR) instrument
368:Cell toxicity, proliferation, and viability
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117:Learn how and when to remove this message
2297:Instruments used in medical laboratories
256:to assess intracellular calcium levels.
1813:Inductively coupled plasma (ICP) device
702:10.2136/sssaj1998.03615995006200020026x
682:Soil Science Society of America Journal
441:European Journal of Medicinal Chemistry
430:
252:measures the fluorescence intensity of
2316:Molecular biology laboratory equipment
1861:Transmission electron microscope (TEM)
612:Environmental Science & Technology
167:. They are widely used in research,
7:
55:adding citations to reliable sources
1856:Scanning electron microscope (SEM)
25:
316:Light scattering and nephelometry
1901:Thermogravimetric analyzer (TGA)
1711:Nuclear magnetic resonance (NMR)
833:"AlphaScreen | BMG LABTECH"
647:Journal of Environmental Quality
519:Journal of Immunological Methods
289:Time-resolved fluorescence (TRF)
31:
42:needs additional citations for
136:PowerWave XS Microplate Reader
1:
811:10.1016/0003-2697(87)90585-9
531:10.1016/0022-1759(83)90303-4
496:10.1016/0003-2697(87)90585-9
453:10.1016/j.ejmech.2019.111981
277:Common applications include
347:Protein–protein interaction
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929:10.1038/s41598-022-24480-3
872:10.1038/s41598-019-54868-7
181:time-resolved fluorescence
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1823:Liquid chromatograph (LC)
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597:10.1080/00103629509369465
566:10.1080/00103629509369298
382:High throughput screening
358:Nucleic acid quantitation
307:Fluorescence polarization
185:fluorescence polarization
1891:Melting-point apparatus
1272:Cryogenic storage dewar
799:Analytical Biochemistry
484:Analytical Biochemistry
1828:Mass spectrometer (MS)
1818:Gas chromatograph (GC)
415:High-content screening
362:Molecular interactions
325:Instruments and assays
254:calcium-sensitive dyes
149:microplate photometers
137:
2203:Acid-resistant gloves
1884:differential scanning
163:events of samples in
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1780:Analytical chemistry
1282:Laminar flow cabinet
988:Laboratory equipment
659:10.2134/jeq2001.2206
591:(15–16): 2519–2529.
51:improve this article
2152:Personal protective
1061:Meker–Fisher burner
921:2022NatSR..1220146S
864:2019NatSR...918425S
694:1998SSASJ..62..473R
624:2000EnST...34.3148T
394:Cellular Uptake of
1995:Function generator
1978:Bench power supply
1917:Analytical balance
1678:Ostwald viscometer
1673:Graduated cylinder
1412:Inoculation needle
909:Scientific Reports
852:Scientific Reports
372:ATP quantification
145:microplate readers
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1124:Mortar and pestle
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632:10.1021/es9912473
618:(15): 3148–3152.
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1121:
1116:
1114:Liquid whistle
1111:
1106:
1100:
1098:
1096:
1095:
1092:
1088:
1085:
1084:
1082:
1081:
1079:Vacuum dry box
1076:
1071:
1066:
1063:
1058:
1053:
1048:
1043:
1041:Heating mantle
1038:
1033:
1028:
1026:Alcohol burner
1022:
1020:
1018:
1017:
1014:
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1003:
1002:
999:
992:
991:
986:
984:
983:
976:
969:
961:
953:
952:
895:
838:
824:
805:(2): 353–360.
789:
768:(2): 318–326.
748:
727:(2): 318–326.
707:
672:
637:
602:
571:
544:
525:(1–2): 55–63.
509:
490:(2): 353–360.
474:
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232:orthophosphate
197:
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169:drug discovery
125:
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66:"Plate reader"
39:
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24:
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10:
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2187:Safety shower
2185:
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2108:
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2097:Wire stripper
2095:
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1927:Spiral plater
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1791:Compositional
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1340:
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1335:
1333:Balance brush
1332:
1330:
1327:
1325:
1322:
1321:
1319:
1315:
1309:Weighing dish
1308:
1306:Weighing boat
1305:
1303:
1300:
1298:
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1198:
1197:Burette clamp
1195:
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1188:
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1057:
1054:
1052:
1049:
1047:
1044:
1042:
1039:
1037:
1034:
1032:
1031:Bunsen burner
1029:
1027:
1024:
1023:
1021:
1015:
1012:
1011:
1008:
1004:
997:
993:
989:
982:
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793:
790:
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781:
776:
771:
767:
763:
762:BioTechniques
759:
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722:
721:BioTechniques
718:
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703:
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629:
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536:
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442:
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431:
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416:
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402:
397:
396:nanoparticles
393:
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367:
364:
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359:
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353:
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315:
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154:
150:
146:
142:
141:Plate readers
135:
131:
121:
118:
110:
99:
96:
92:
89:
85:
82:
78:
75:
71:
68: –
67:
63:
62:Find sources:
56:
52:
46:
45:
40:This article
38:
34:
29:
28:
19:
18:Plate readers
2258:Fire blanket
2196:Eye and hand
2182:Rubber apron
2046:Oscilloscope
2010:Potentiostat
1937:Plate reader
1936:
1833:pH indicator
1803:CHN analyzer
1798:AutoAnalyzer
1591:Round-bottom
1484:Boston round
1347:Filter paper
1302:Refrigerator
1219:Retort stand
1187:Clamp holder
1183:Beaker clamp
1149:Vortex mixer
1144:Stirring rod
1139:Static mixer
1069:Teclu burner
915:(1): 20146.
912:
908:
898:
858:(1): 18425.
855:
851:
841:
827:
802:
798:
792:
765:
761:
751:
724:
720:
710:
685:
681:
675:
650:
646:
640:
615:
611:
605:
588:
584:
574:
557:
553:
547:
522:
518:
512:
487:
483:
477:
444:
440:
433:
400:
377:Immunoassays
339:Protein and
328:
319:
310:
292:
276:
263:
260:Luminescence
241:
238:Fluorescence
211:colorimetric
199:
177:luminescence
173:fluorescence
148:
144:
140:
139:
113:
104:
94:
87:
80:
73:
61:
49:Please help
44:verification
41:
2246:Other items
2172:Face shield
2019:Measurement
2000:Galvanostat
1960:Electronics
1910:Other items
1896:Thermometer
1879:Calorimeter
1808:Colorimeter
1752:Gas syringe
1735:Other items
1663:Eye dropper
1538:Watch glass
1523:Evaporating
1502:Cold finger
1317:Other items
1223:Screw clamp
1215:Pinch clamp
1204:Flask clamp
1154:Wash bottle
1109:Homogenizer
387:Bead-based
341:cell growth
296:lanthanides
175:intensity,
2177:Respirator
2118:Test probe
2036:Multimeter
1848:Microscopy
1668:Eudiometer
1632:Separatory
1601:Volumetric
1566:Erlenmeyer
1494:Condensers
1458:Dean–Stark
1407:Wire brush
1367:Microscope
1362:Centrifuge
1337:Cork borer
1292:Petri dish
1267:Agar plate
1254:Containers
1237:Wire gauze
1074:Water bath
1036:Desiccator
688:(2): 473.
447:: 111981.
425:References
279:luciferase
196:Absorbance
153:biological
107:April 2012
77:newspapers
2268:Fume hood
2213:Glove box
2066:Voltmeter
1451:Apparatus
1440:Glassware
1329:Autoclave
1324:Aspirator
1277:Incubator
1211:Iron ring
1134:Sonicator
1104:Chemostat
1046:Hot plate
469:210892159
206:MTT assay
2310:Category
2167:Lab coat
2092:Tweezers
2082:Heat gun
1838:pH meter
1747:Bell jar
1627:Dropping
1581:Florence
1571:Fernbach
1533:Syracuse
1392:Scoopula
1342:Crucible
1051:Lab oven
947:36418509
890:31804555
784:10023544
743:10023544
667:11790034
461:31978780
352:Reporter
216:ammonium
161:physical
157:chemical
2106:General
2026:Ammeter
1726:Thistle
1683:Pipette
1658:Cuvette
1648:Burette
1617:BĂĽchner
1610:Funnels
1596:Schlenk
1576:Fleaker
1556:BĂĽchner
1477:Bottles
1397:Spatula
1387:Stopper
1357:Forceps
1257:Storage
1174:Holders
1094:Shakers
1065:Striker
1013:Heaters
1000:General
938:9684140
917:Bibcode
881:6895130
860:Bibcode
819:3434778
690:Bibcode
620:Bibcode
539:6606682
504:3434778
406:ELISPOT
389:epitope
224:nitrite
220:nitrate
191:Methods
91:scholar
2140:Safety
1742:Beaker
1721:Thiele
1706:Cragie
1701:Drying
1622:Hirsch
1586:Retort
1548:Flasks
1516:Dishes
1507:Liebig
1468:Kipp's
1382:Splint
1192:Tripod
1170:Clamps
1166:Stands
1129:Shaker
1091:Mixers
1016:Dryers
945:
935:
888:
878:
817:
782:
741:
665:
537:
502:
467:
459:
354:assays
343:assays
335:ELISAs
183:, and
134:BioTek
93:
86:
79:
72:
64:
2075:Tools
1693:Tubes
1528:Petri
465:S2CID
391:assay
202:ELISA
98:JSTOR
84:books
1757:Vial
1716:Test
1352:File
1056:Kiln
943:PMID
886:PMID
815:PMID
780:PMID
739:PMID
663:PMID
535:PMID
500:PMID
457:PMID
300:FRET
228:urea
70:news
933:PMC
925:doi
876:PMC
868:doi
807:doi
803:166
770:doi
729:doi
698:doi
655:doi
628:doi
593:doi
562:doi
527:doi
492:doi
488:166
449:doi
445:189
283:ATP
267:PMT
159:or
147:or
53:by
2312::
941:.
931:.
923:.
913:12
911:.
907:.
884:.
874:.
866:.
854:.
850:.
813:.
801:.
778:.
766:26
764:.
760:.
737:.
725:26
723:.
719:.
696:.
686:62
684:.
661:.
651:30
649:.
626:.
616:34
614:.
589:26
587:.
583:.
558:26
556:.
533:.
523:65
521:.
498:.
486:.
463:.
455:.
443:.
285:.
226:,
222:,
218:,
187:.
179:,
155:,
980:e
973:t
966:v
949:.
927::
919::
892:.
870::
862::
856:9
835:.
821:.
809::
786:.
772::
745:.
731::
704:.
700::
692::
669:.
657::
634:.
630::
622::
599:.
595::
568:.
564::
541:.
529::
506:.
494::
471:.
451::
120:)
114:(
109:)
105:(
95:·
88:·
81:·
74:·
47:.
20:)
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