This guide provides important information to help you make the right decision. Learn about the different types of readers/detectors; read modes, applications, technologies and other important considerations.
Microplate readers are used to detect biological, chemical or physical events in microtiter plates via the measurement of light. Microplate readers/detectors are the key workhorses in many laboratories and are used extensively for many applications across a wide range of disciplines including life sciences, drug discovery, bioassay validation, quality control, drug safety, toxicity testing, clinical diagnostics and biopharmaceutical/pharmaceutical manufacturing processes.
Although a well-established product category, microplate readers continue to evolve towards greater functionality, flexibility, speed and throughput. Currently, there are a wide variety of microplate readers available, offering different capabilities and functionalities.
Microplate readers differ by the type of detection mode. The most common detection modes are absorbance, fluorescence and luminescence. Additional detection modes include fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF), fluorescence polarization, bioluminescence resonance energy transfer (BRET), AlphaScreen and nephelometry.
Microplate readers also differ by detector technology. Light detection can be performed using filters, monochromators or spectrophotometry. Monochromator based technology has the most flexibility in wavelength detection, so is most suitable for labs with variable detection requirements. Newer spectrometer-based readers can capture an entire spectrum of wavelengths, offering even more flexibility in detection. Filters are for fixed wavelength detection but offer greater sensitivity, so are more appropriate for labs with limited detection needs. Hybrid systems combining these technologies are also available. In addition, lasers rather than flash lamps are better for time-resolved fluorescence and AlphaScreen detection.
Microplate readers can be purchased as either single-mode or multimode; the latter combines several read modes in one instrument. Multimode microplate readers enable researchers to perform multiple assay types in one system. Multimode readers are typically more expensive than single-mode readers but purchasing one multimode reader is likely to be more cost-effective than buying several dedicated machines. In addition, they do not require much more bench space than a single reader and are often modular and upgradable, allowing a laboratory to purchase only what they need at the time but allowing for future additions.
For example, the PHERAstar® FSX, (see Figure 1) from BMG LABTECH is a multimode reader, which includes a Simultaneous Dual Emission detection system that allows two wavelengths to be read at the same time. The sensitivity of the reader, combined with faster processing times, make it perfect for more sophisticated applications, including FRET, TR-FRET and BRET assays. This modular reader is also compatible with a number of accessories designed to enhance plate reading workflow, including stackers and incubators. Watch the video (Figure 1) to hear Senior Scientist, Helen Harrison, describe how the PHERAstar® FSX has accelerated workflow at Bicycle Therapeutics Ltd.
Figure 1: The PHERAstar® FSX, from BMG LABTECH
When choosing a microplate reader/detector, the first, and perhaps the most important, considerations are the application requirements. Microplate readers are used across diverse scientific disciplines for varying applications the most common methods are briefly introduced below.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is an immunodetection assay that uses labelled secondary antibodies to detect specific antigens. The secondary antibody conjugate, such as horseradish peroxidase (HRP), is detected via an enzyme-mediated chromogenic change. ELISAs are used extensively in the life sciences, pre-clinical research and clinical diagnostics. The majority of plate readers are able to read ELISAs.
Nucleic Acid Quantification
DNA and RNA absorb light within the UV range. Absorbance of a sample at 260 nm can be used to calculate the concentration of double-stranded DNA, single-stranded DNA or single-stranded RNA. Protein contamination, common in DNA and RNA preparations, can be determined using the 260/280 nm absorbance ratio. Plate readers with UV bandwidth detection capabilities are suitable for this detection. The new Trinean DropSense96 content analysis platform is designed for fast quantification of DNA, RNA and protein concentration from just 2μl of undiluted sample. Watch the video (Figure 2) to learn more.
Figure 2: The Trinean DropSense96 content analysis platform
Reporter genes, such as luciferase or GFP, can also be assessed in microplate readers, enabling in vitro and in vivo determination of gene expression for studies using markers of genetic alteration. Any reader with appropriate wavelength detectors can be used for this detection.
Measurement of enzyme activity is a common assay in life sciences and pre-clinical research. The time-resolved, enzyme-dependent accumulation of a marker or product can be used to determine enzyme kinetics. The majority of plate readers have this capability.
Basic Protein Assays
Protein quantitation is another common application. As discussed above, protein concentration can be determined by absorbance at 280 nm, however, many chromogenic reagent-based assays have also been developed. Microplate readers can be used to measure total protein concentrations using a variety of commercially available kits.
Fluorescent Protein Assays
Fluorescent markers such as EGFP, YFP, mCherry and mTomato can be used to measure a variety of real-time cell-based activities, including: intracellular transport, protein signaling, receptor desensitization, migration, division, apoptosis, metabolism, differentiation, chemotaxis, transcription and translation. These assays are often applied to live cells, necessitating a microplate reader with appropriate environmental controls. BMG LABTECH’s CLARIOstar® comes with oxygen and carbon dioxide controls to support live cell assays (see Figure 3).
Figure 3: The CLARIOstar®, from BMG LABTECH
Fluorescent or luminescent markers are also used in pharmacological and drug discovery assays. These are used to investigate ligand binding and protein interactions. TTP Labtech’s ameon® lifetime reader incorporates fluorescence lifetime technology, enabling cost-effective screening of common drug targets as well as challenging epigenetic targets. Read about a novel nanoBRET assay developed in association with BMG LABTECH and watch the video (see Figure 4) to hear Dr Kevin Pfleger, Associate Professor, University of Western Australia, describe how BMG LABTECH’s CLARIOstar® and PHERAstar® FSX were instrumental in its development.
Figure 4: Dr Kevin Pfleger discusses nanoBRET assay development
Cell Density & Counting
Bacterial cell counting can be determined by optical density at 600 nm, which indicates the growth phase for harvesting the cells. For other cell counting applications, discover how the Spark® 20M Multimode Microplate Reader (Figure 5), from Tecan, can optimize cell incubation, counting and dispensing functions, combined with automated cell imaging and confluence measurement, for long-term analysis.
Figure 5: The Spark™ 20M Multimode Microplate Reader, from Tecan
Cell Viability, Proliferation and Cell Death
There are many different commercially available cell-based assay kits that enable cell viability, proliferation and apoptosis to be measured. These include those for monitoring ATP, measuring caspase activity and detecting bromodeoxyuridine (BrdU). They are also used in toxicity testing in pre-clinical drug development.
High Content Screening
Drug discovery & manufacturing applications often require high content screening (HCS) and/or high throughput screening (HTS). Most of the applications above can be conducted in high content, multi-well plates in microplate readers. High throughput drug screening for target validation and ADMET (absorption, distribution, metabolism, elimination and toxicity) is a key process in drug discovery. However, phenotypic cell-based assays are now becoming more commonplace. In addition to conventional assays, these readers enable phenotypic screening of cells, either by well or per cell. Multimode microplate readers have been designed specifically for this HTS combined with imaging. Examples include the ImageXpress Micro 4 High-Content Imaging System from Molecular Devices and the Operetta CLS™ High-Content Analysis System from PerkinElmer, Inc. (Figure 6).
Figure 6: The Operetta CLS™ High-Content Analysis System from PerkinElmer, Inc.
Phenotypic screening is used routinely in drug discovery for the identification of substances that alter the phenotype of a cell or an organism. The target cells are screened with compounds or biopharmaceuticals to assess modulation of the activity of interest. In vitro phenotypic screening uses cell lines and cell based assays and is often performed in high content microplates, and may utilize live cells. Watch the video (Figure 7) to find out how the Corning Epic® System is enabling high throughput assay miniaturization at AstraZeneca from Senior Scientist, Helen Plant.
Figure 7: The Epic® System, from Corning at AstraZeneca
Live Cell Assays
In live, real-time cell based experiments, it is beneficial to read from the bottom of the microplate and rather than the top. Bottom reading offers several advantages for cell based detection: the light collector can be placed closer to the sample and the cell layer adherent closer to the bottom of the well, which decreases light dissipation. Moreover, the interfering effect of the cell culture medium is significantly reduced. Both factors positively affect sensitivity. In addition, bottom reading allows for a cover or lid to be placed on top of the microplate to prevent cell contamination and liquid evaporation. This is particularly important in time-lapse experiments. Several readers are optimized for live cell assays, including BMG LABTECH’s PHERAstar® FSX (see Figure 1) and CLARIOstar® (see Figure 3) readers, which use a proprietary Direct Optic Reading system to eliminate the need for fiber optics. BioTek Instruments, Inc.’s new Lionheart™ FX Automated Live Cell Imager features brightfield, color brightfield, phase contrast and fluorescence channels for maximum support for a wide range of imaging applications, find out more in the video (Figure 8). Plus, watch the SelectScience webinar to learn how multimode microplate reader technology from Tecan is automating cell migration and wound healing assays.
Figure 8: The Lionheart™ FX Automated Live Cell Imager from BioTek Instruments, Inc.
3D Cell Culture Assays
Recently, there has been increased drive to develop assays and readers for 3D cell cultures. These spheroids or cell clumps can be grown in multi-well plates and more closely mimic the endogenous environment, which is important in both life sciences and drug discovery applications. Watch the video (Figure 9) with Kirk McManus, Associate Professor, University of Manitoba, and Senior Scientist, CancerCare Manitoba, to hear how an automated workflow incorporating BioTek Instruments, Inc.’s BioSpa™ 8 Automated Incubator and Cytation 3 Cell Imaging Multi-Mode Reader, is being used for 3D tumor spheroid assays to help to advance cancer research.
Figure 9: Kirk McManus, University of Manitoba and CancerCare Manitoba, discusses a high-throughput workflow for phenotypic screening
Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen)
AlphaScreen is a versatile assay technology developed to measure analytes using a homogenous protocol that enables sensitive and precise interrogation of signaling pathways, receptors, enzymes and kinase targets, in a cell-based format. AlphaScreen has been particularly useful for screening GPCRs, growth factor receptors, intracellular MAPK inhibitors and many other signaling pathways.
Homogeneous Time Resolved Fluorescence (HTRF)
Utilizing rare-earth lanthanides with long emission half-lives as donor fluorophores, HTRF technology combines standard FRET with the time-resolved measurement (TR) of fluorescence. HTRF is commonly used for GPCR and kinase screening, two of the most important target classes investigated within drug discovery. Other HTRF applications include discovery of new biomarkers, studies of protein-protein interactions, epigenetics and an alternative method for bioprocess monitoring.
Microplate readers/detectors are used for numerous applications and, as technologies evolve, even more applications will emerge. When purchasing a microplate reader/detector, it is important to establish what applications you will be using it for now, and in the future. You will want to ensure that your chosen microplate reader/detector is capable of fulfilling all your requirements but also consider how easily upgradable it is for your future needs.
Try it Out: Once you have considered both your application needs and the different technologies that are available, the chances are you will have narrowed down the choice to just a few microplate readers. Before going ahead and purchasing a microplate reader/detector, it is highly recommended that you try it out. Most manufacturers will let you demo the instrument for a trial period. This is a great opportunity to ensure the reader fulfills all your requirements, is easy to use and to make sure you get the right support and training to use the instrument.
Technical Support and Warranty: Consider the extent of the technical support and training that is available from the manufacturer at the point on purchase, is it included in the purchase cost? It is also recommended that you look into the company’s policy on after-sales service and standard warranty. While everything is operating well, this may be the last thing on your mind, but in the event that after-sales support is required, it is important to know how accessible that is.
Accessories and Add-Ons: Not all microplate readers have the same standard features. Be sure to ask what is included with the reader at the time of demonstration. Optional items may be key parts of the kit, including: computers to run the instrument, filters or filter cubes, software licenses, software upgrades, FDA compliant software, environmental controls or injectors.
Software: Microplate readers take hundreds or thousands of measurements, so data acquisition and analysis are key to efficient workflow. Make sure that you know how to use the software for your current needs and how to keep it updated, find out how well it is supported by the manufacturer.
Flexibility: Many labs have equipment from numerous different manufacturers. Consider how well your chosen reader will integrate with your existing equipment and how easy it will be to upgrade in the future. Many models have modular design and add-ons but their compatibility may be brand limited.
Future Needs: Taking into consideration current and future trends is essential in maximizing the lifespan of the microplate reader/detector. As systems become more sophisticated, integration, convenience and application specificity will be fundamental. Systems will become faster and more efficient and be able to run a larger variety of different assays. Application possibilities will increase as manufacturers work to provide solutions to individual and industry demands. Manufacturers are taking customer feedback and requirements into consideration, with many developing strategies for even greater specificity assays and technology for the future.
Whether purchasing a microplate reader/detector for a new application, or replacing an existing system, there are a number of factors to consider. You will need to examine your current and future application needs and determine which of the available technologies best suits these applications.
Visit the Microplate Readers / Detectors product directory for an overview of the latest products from leading manufacturers. Keep up-to-date with the latest techniques and advances in technology by visiting the SelectScience Microplate Readers / Detectors pages for application notes, videos and the latest news.