Microplate readers are used to detect biological, chemical or physical events in microtiter plates via the measurement of light. Microplate readers are the key workhorses in many laboratories and are extensively used for many applications across a wide range of disciplines including life sciences, drug discovery, bioassay validation, quality control and biopharmaceutical manufacturing processes.

Although a well established product category, microplate readers are continuing to evolve towards greater functionality, flexibility, speed and throughput. Currently there are 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) and nephelometry.

Microplate readers can be purchased as either single-mode or multimode, where the latter combines several read modes on one instrument. Multimode microplate readers enable researchers to perform multiple assay types in one system. Inevitably 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.

When choosing a microplate reader the first consideration, and perhaps the most important, is what applications is it going to be used for? Microplate readers are used for numerous applications across a wide range of disciplines.

If you work within a life science laboratory, you may be using a microplate reader for applications such as:

• Enzyme-Linked Immune-Absorbent Assay: ELISA is an immunodetection assay which allows for identification of specific antigens in samples. Horseradish peroxidise (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 tetramethybenzidine hydrochloride (TMB), which absorbs visible light at 450 nm, is used to detect binding.
• 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.
• 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 are typically used that change color linearly in response to the amount of protein in the sample. Commonly used assays include bicinchoninic acid (BCA), Lowry and Bradford.
• Cell Density: An important aspect of preparing bacterial cultures is to determine the growth phase before harvesting the cells. Measurement at 600 nm allows for this determination.
• 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).
• 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.

If you work in a drug discovery laboratory, high throughput screening (HTS) of compounds and targets is likely to be a key application area. For such applications speed and sensitivity are of major importance. Consequently several multimode microplate readers have been designed specifically for HTS applications. The ability to measure all wells of a microplate at once is a factor to consider alongside reliability, sensitivity and consistency, which are essential for high throughput.

There are several assay technologies that have been developed specifically for screening applications, which include:

• AlphaScreen: AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is 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.
• HTRF: Utilizing rare-earth lanthanides with long emission half-lives as donor fluorophores, Homogeneous Time Resolved Fluorescence (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 and an alternative method for bioprocess monitoring.

Microplate readers are used for many more applications and as technologies evolve even more applications will emerge. When purchasing a microplate reader is important to establish what applications you will be using it for. You will need to ensure that your chosen microplate reader 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.

The type of technology incorporated into a microplate reader is what differentiates one from another. It is therefore necessary to determine what technology you require for your anticipated applications. This section reviews some of the key technological features that should be considered when purchasing a microplate reader.

Monochromator v. Filters

Multimode microplate readers are typically built with a focus on fluorescent detection as it is the most diverse, complex, and common read mode used in biotechnology applications. Scientists purchasing a multimode microplate reader for fluorescent detection will need to choose between two types of system for wavelength selection. The choice between a filter-based reader and monochromator-based reader will have a fundamental impact on the applications that the instrument can perform.

Monochromators use diffraction gratings to physically separate the individual wavelengths present in the white light coming from the instrument’s light source. A series of slits allow for selecting a specific wavelength to excite the sample. Monochromator-based readers offer several benefits over filter-based readers:

• They are very convenient to use and highly flexible. Wavelengths can be easily selected through the software and are generally available in 1 nm increments. Manual manipulation of the system and storage of additional accessories are not required. This also means that new applications can be accommodated with relative ease and low additional cost.
• Monochromators can run spectral scans that are used to characterize new fluorophores or study spectral shifts in some assays, although they exhibit higher background noise than comparable cuvette-based readers.

Monochromator-based readers do also have several disadvantages. The inherent design of the diffraction gratings causes significant loss of light both to and from the sample, which decreases overall sensitivity. Another disadvantage is the higher cost associated with the units, especially when compared to the performance in common applications.

Optical filters are characterized with a central wavelength and a bandwidth. These two fixed parameters precisely define which wavelengths are going to the sample on the excitation side and from the sample to the detector on the emission side. Filter-based microplate readers also have a number of important advantages:

• Filter-based readers are typically less expensive than monochromator-based microplate readers. Filter wheels or slides are relatively cheap compared to the parts required for a monochromator, and the light source required to produce the same level of sensitivity does not need to be as powerful.
• A filter-based reader is more effective at delivering light to the sample and light blocking between the excitation and emission channels for superior sensitivity.
• Filter-based systems permit bandwidth selection. Bandwidth selection is beneficial as a filter can be dedicated to a specific assay for maximum sensitivity and can have a bandwidth from a few nanometers to greater than 100 nm, which is necessary for low-level fluorescence assays.
• Filter-based microplate readers can rapidly switch between two wavelengths while monochromator-based systems are typically much slower.

The main disadvantage of filter-based microplate readers is that separate filter sets must be purchased and maintained for different applications. Additionally, the fixed wavelength of a filter also disallows the use of spectral scanning applications.

Currently the majority of microplate readers are either monochromator- or filter-based. However, some manufacturers have found a way to incorporate both technologies. In the future, such instruments are likely to fill a large gap in the microplate reader market.

Speed and Throughput

The speed and throughput that you require from your microplate reader will largely depend on the application. If you are analyzing a large number of samples per day the speed, or read time per plate, of the microplate reader becomes an important factor. Many instruments now incorporate multiple detectors, which can dramatically increase the speed of the system. For example fluorescence polarization, FRET and BRET experiments require two emission wavelengths to be measured. Some microplate readers will measure the plate twice, but those that contain multiple detectors will only need to measure the plate once, which saves time and decreases measurement variability.

The most common microplate format used in academic research laboratories or clinical diagnostics laboratories is 96-well, with a typical reaction volume between 100 and 200 µL per well. Higher density microplates (384- or 1536-well) are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. The majority of microplate readers can handle 96- and 384-well microplates, but higher densities may require a more specialized instrument. Although not covered in this guide, choosing the correct microplate for your application can mean the difference between indifferent and accurate results and should therefore be selected carefully.

Software

When purchasing a microplate reader it is imperative to consider the ease-of-use of the system. This is in part determined by the by the system’s software. It is important to evaluate the software thoroughly, ensure it is intuitive and has a user friendly interface.

In addition to controlling the microplate reader, the software is likely to be used for data analysis. You should look for an instrument with a software package that enables you to get up and running in a short period of time, but make sure the software has expanded capabilities so you can perform more complex analyses in the future. You should also investigate how often the software is upgraded and whether you will be eligible for such upgrades. Furthermore, if there are multiple users, it is also important to ask whether how many computers the license covers, as you may want to analyze the data on a separate machine.

Flexibility

When choosing a microplate reader it is important to determine whether it reads plates from the top, bottom or both. Whether you require top reading or bottom reading will depend on your applications. For example, for cell-based assays, it is better to read from beneath the microplate. Most cells, particularly adherent cells, will grow on the bottom of the microplate. In addition, reading from the bottom allows for a cover or lid to be placed on top of the microplate to prevent cell contamination and liquid evaporation. Top reading usually provides better signal-to-noise ratios, which may be useful for solution-based assays such as DNA or protein quantification for example.

It is important to note that many manufacturers offer microplate reader accessories, which may enable you to broaden your current applications. For cell-based assays for example, you may wish to control the external environment. Some microplate readers have a gas vent, which can be used to purge the microplate chamber with different gases and may be relevant if you wish to measure cell growth in anaerobic conditions for example. Many microplate readers have built in incubators and shakers, which can be particularly useful when performing cellular growth assays.

Many of today’s popular assays, for example enzyme kinetics, calcium flux, dual luciferase, and ORAC, require a signal to be monitored before and after the addition of a reagent. If you envisage requiring such a function you will need a microplate reader that is capable of reagent injection during your experiment. Many of these assays produce signals that decay very quickly after reagent addition, becoming unreadable in only a few seconds. Therefore your microplate reader must be able to inject into a well of a microplate and then be able to quickly or instantly measure that well after injection.

Upgrading Your Microplate Reader

The option to upgrade an instrument is also a valid factor to consider. Many of the microplate readers on the market today are modular in design, enabling them to grow as requirements change. Upgrading your microplate reader will typically involve the incorporation of new detection modes. The selection of a new microplate reader is largely driven by budget and the ability to upgrade allows a lower cost of entry without restricting future applications. You should discuss with the manufacturer how easy it is to upgrade its instruments.

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 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.

When purchasing a microplate reader, like with any major piece of laboratory equipment, in addition to considering your budget there are several other general considerations that should not be overlooked. For example, you should 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. 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.

Taking into consideration current and future trends is essential in maximizing the lifespan of the microplate reader. 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 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 product directory for an overview of the latest microplate readers from leading manufacturers and read hundreds of user reviews. Keep up to date with the latest microplate reader techniques by visiting the SelectScience application note and video libraries.