What should you think about when considering a microplate reader or microplate detector purchase for your laboratory? Perhaps you are looking to upgrade for a specific capability, application or mode. Maybe you are a first-time buyer of a microplate reader/detector and need to know what will be useful in your work. 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 extensively used for many applications across a wide range of disciplines including life sciences, basic research, target discovery, lead discovery, bioassay validation, quality control, drug safety, toxicity testing and biopharmaceutical/pharmaceutical manufacturing processes.
Although a well established product category, microplate readers are continuing to evolve towards greater functionality, flexibility, speed and throughput. Currently there is a wide variety of microplate readers on the market, offering different capabilities and functionalities.
Microplate readers largely differ by the type of detection mode they offer. Common detection modes include absorbance, fluorescence and luminescence. Microplate readers may also include additional detection modules such as those for fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF) , the variant TR-FRET, fluorescence polarization, bioluminescence resonance energy transfer (BRET), AlphaScreen and nephelometry.
Microplate readers also differ by the technology used in these detection modes. Absorbance measurements can be performed with filters, monochromators or a spectrometer. Filter- and monochromator-based instruments capture one wavelength at a time, while the newer spectrometer-based readers capture an entire spectrum of wavelengths. Fluorescence measurements can be performed using filters or monochromators. Filters offer great sensitivity, while monochromators offer great flexibility, and new linear variable filter monochromators offer the best of both. Lastly, lasers instead of a flash lamp can be used for better performance in time-resolved fluorescence and AlphaScreen measurements. Just be aware that different technologies may be used for the different detection modes in the different microplate readers.
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 consume 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. For example, the CLARIOstar®, from BMG Labtech, has recently been improved with a new Atmospheric Control Unit (ACU) allowing for independent, simultaneous control of both O2 and CO2 within the microplate reader chamber. This new ACU improvement provides the optimal environment for any live cell based assay, from standard cell growth to hypoxic or cytotoxicity assays.
When choosing a microplate reader/detector the first, and perhaps the most important, consideration is what application(s) will be measured? Microplate readers are used for numerous applications across a wide range of disciplines.
If you work within life science or basic research, you may be using a microplate reader for many varying applications. Some of these applications are also used in drug discovery for pre-clinical testing and toxicity.
Research-based applicationsEnzyme-Linked Immunosorbent Assay (ELISA)
ELISA is an immunodetection assay that allows for identification of specific antigens in samples. Horseradish peroxidase (HRP) is typically conjugated to the secondary antibody to detect the binding of antibody to the antigen of interest. A chromogenic substrate for HRP, such as tetramethylbenzidine dihydrochloride (TMB), which absorbs visible light at 450 nm, is used to detect binding. ELISA is used widely in life sciences but also in pre-clinical testing for drug development.Protein and Nucleic acid Quantification
DNA and RNA absorb light efficiently in the UV range. Quantitation of nucleic acid occurs at 260 nm. Purity can be determined by measuring both at 260 nm and 280 nm, as protein absorbs at 280 nm. Protein is a common contaminate in DNA and RNA preparations. The SpectraMax® M5e Microplate Reader, from Molecular Devices, uses temperature-independent normalization corrections for varying well volumes and can eliminate standard curves by allowing the calculation of concentrations directly from the absorbance with a known extinction coefficient; a known issue in nucleic acid and protein quantitation.Kinetics
Enzyme kinetics is an important aspect of enzymology. The use of a chromagenic substrate allows for the determination of enzyme kinetics such as the Michaelis-Menten constant and the rate of a reaction.Protein Assays
A variety of commercially available kits allow for measuring the amount of total protein in a sample. Reagents that change color linearly in response to the amount of protein in the sample are typically used. Commonly used assays include bicinchoninic acid (BCA), Lowry and Bradford.Cell Density
Determining the growth phase before harvesting the cells is an important aspect of preparing bacterial cultures. Measurement of optical density (O.D.) at 600 nm allows for this determination. The Spark™ 10M Multimode Microplate Reader, from Tecan, has dedicated cell modules, such as the CellChip, and Fusion Optics for cell counting. The new Cytation™ 5 Cell Imaging Multi-Mode Reader (see Figure 1), from BioTek, is a uniquely integrated, configurable system that combines automated digital widefield microscopy with conventional multi-mode microplate detection to provide phenotypic cellular information and well-based quantitative data. A good example of a cell density study can be seen in this application, in which the Cytation™ 5 is used to evaluate the use of a high density cell exclusion assay in 384-well format for investigation of cell migration using a cell line stably expressing green fluorescent protein (GFP). Images were captured by the Cytation™ 5 and analyzed using automated digital wide field fluorescence microscopy.
Figure 1: The new Cytation™ 5 Cell Imaging Multi-Mode Reader, from BioTekCell 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. Cell based assays are crucial in the early stages of disease research and identification of potential protein pathways. TTP Labtech’s acumen® Cellista Laser Scanning Imaging Cytometer can rapidly and accurately quantify cell colony formation in soft agar, as described in this application note. This laser scanning imaging cytometer allows the operator to scan whole wells to enumerate fluorescent colonies and can also be used to determine the size of colonies through the application of a spherical volume algorithm.Fluorescent Protein Assays
EGFP, YFP, mCherry, mTomato and the many other variants of GFP can be used to measure a variety of real-time cellular-based activities, including: intracellular transport, protein signaling, receptor desensitization, cell movement, migration, division, apoptosis, metabolism, differentiation, chemotaxis, transcription and translation. This application note presents an interesting example of a fast, simple, transient assay using BMG LABTECH’s CLARIOstar® that can be used to measure GFP and mCherry fluorescence in living protoplasts in a microplate format.
Figure 2: The CLARIOstar®, from BMG Labtech, a new Atmospheric Control Unit (ACU)Gene Expression
Reporter genes, such as luciferase, are important tools for studying gene expression. The use of reporter genes allows the in vitro and in vivo measurement of gene expression from virtually any endogenous genetic control element. Luciferase enzyme and its subsequent luminescent reaction is often the gene reporter of choice for many experimental conditions.
Drug Discovery Based Applications
If you work in a target or lead discovery laboratory, high content screening (HCS) of protein targets and high throughput screening (HTS) of compounds are likely to be a key application areas. Of the most exciting of these screening applications in the modern research or screening lab is cell based assays.
Cell based assays are used in more than half of all high-throughput drug screenings for target validation and ADMET (absorption, distribution, metabolism, elimination and toxicity) in the early stages and pre-clinical stages of drug discovery. Most recently, the technique of using cell based assays in phenotypic approaches, alongside traditional target-based methods, help to ensure the physiological relevance of results early in the drug discovery process. Live-cell assays are increasingly used for phenotypic screening; this will be discussed in more detail below. For screening, speed and sensitivity are of major importance. Consequently, several multimode microplate readers have been designed specifically for HTS and cell-imaging applications, such as the EnSight™ Multimode Plate Reader, from PerkinElmer, see Figure 3 below.
Figure 3: The EnSight™ Multimode Plate Reader, from PerkinElmer
In this example, the quality control of cell based assays is determined with cytometric parameters using the brightfield, digital phase and fluorescent imaging modes on PerkinElmer’s EnSight™ Multimode Plate Reader.Live-Cell Assays
In live, real-time cell based experiments, it is beneficial to read from the bottom of the microplate and not from the top. Bottom reading offers several advantages for cell based detection: the light collector can be placed closer to the sample, the cell layer adherent to the bottom of the well, decreasing 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.
In an effort to perfect cell based assays in microplate readers, new technology has eliminated the need for fiber optics both in top and bottom reading. BMG LABTECH uses its proprietary Direct Optic Reading system, in the PHERAstar FS® reader and the CLARIOstar®. Just like a microscope, the PHERAstar FS® and CLARIOstar® take advantage of a free-air optical path to direct and focus light on to either the bottom or top of the microplate, no fiber optics are used. This is achieved by a series of software-controlled, motor-driven mirrors. The Direct Optic Reading system displayed a significant improvement in signal-to-blank ratios when compared to readers with fiber optics.
The Spark™ 10M Multimode Microplate Reader, from Tecan, also has new technology for automated live-cell analysis.
Figure 4: The Spark™ 10M Multimode Microplate Reader, from TecanPhenotypic Screening
Phenotypic screening is a type of screening used in life science or basic research and drug discovery to identify substances such as small molecules, peptides, or RNAi that alter the phenotype of a cell or an organism in a desired manner. It is used in the pharmaceutical industry particularly to develop a hypothesis that a certain biological target is disease modifying, and then can screen for compounds or biopharmaceuticals that modulate the activity of this purified target. Phenotypic screening can be performed either in vitro, the simplest method using cell lines and assays that measure cell density and cell death, or in vivo usually at pre-clinical stage, for testing/evaluation of a pharmaceutical for potential therapeutic benefit across many different types of animal models representing different disease states.
This poster describes the utility of the acumen® Cellista Laser Scanning Imaging Cytometer, from TTP Labtech, for practical high-throughput, full deck phenotypic screening in capacities of over 400,000 wells/day, with the ability to then automatically run secondary screening in an imaging mode. Watch scientists discuss the phenotypic screening capabilities of the EnSight™ Multimode Plate Reader, from PerkinElmer, and how they use phenotypic screening in their research in this video.
There are several assay technologies that have been developed specifically for screening applications, which include:Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen)
AlphaScreen is a versatile assay technology developed to measure analytes using a homogenous protocol. It enables the highly sensitive and precise interrogation of various signaling pathways, receptors and kinase targets, and the measurement of full-length, endogenous protein phosphorylation in a cell based format. A wide variety of applications for AlphaScreen have been reported. It is especially ideal for screening GPCRs and growth factor receptors, and for screening intracellular kinase inhibitors of MAPK and 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 many more applications and as technologies evolve, even more applications will emerge. When purchasing a microplate reader/detector, is important to establish what applications you will be using it for. You will need to ensure that your chosen microplate reader/detector is capable of fulfilling all of your requirements. In addition, you will need to consider any future applications and the needs of any other users of the instrument.
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 of time and it is important to use this time wisely to ensure the instrument is capable of fulfilling all of your requirements.
Technical Support and Warranty: Consider the extent of the technical support and training that is available from the manufacturer at the point on purchase. 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 will this be achieved.
Is it included with the Reader? Not all microplate readers have the same standard features. Make sure to ask what is included with the reader at the time of demonstration. Optional items can include: computers to run the instrument, filters or filter cubes, software licenses, software upgrades, FDA compliant software or injectors.
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, such as those currently being requested in the food/beverage and biofuels industries, as well as in emerging countries. Manufacturers are taking into consideration customer feedback and requirements, 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 SelectScience Microplate Readers / Detectors product and user product reviews directory for an overview of the latest products from leading manufacturers. Keep up-to-date with the latest techniques and advances in Microplate Reader technology by visiting the SelectScience Microplate Readers / Detectors pages for application notes, videos and the latest news.
“TTP Labtech has excellent after-sales care. The customer-vendor relationship continues well beyond the purchase, and the support is fast and efficient.”
Sean Johnston, Harvard Medical School
"It is very easy to use and produces high quality images.”
Sunil Rangarajan, University of Alabama at Birmingham