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Please check out our Microplate Reader / Microtiter Plate Reader section for more information or to find manufacturers that sell these products
Some pieces of laboratory hardware are ubiquitous. Pipettors are one example, and microcentrifuges another. Then there are microplate readers.
Used for everything from enzyme-linked immunosorbent assays (ELISAs) to NADH quantitation, protein–protein interaction detection to testing wine, microplate readers are the sine qua non of medium- and high-throughput laboratories across a range of disciplines, including chemistry, drug development, and proteomics.
For clarification, a microplate reader is not a specific kind of instrument; the term refers to any instrument that can load an SBS-formatted microplate and read it. Several reading modes are available, most typically absorbance, fluorescence, and luminescence, but also including more specialty modes such as time-resolved fluorescence; fluorescence polarization; fluorescence resonance energy transfer (FRET); and nephelometry, a light-scattering detection method commonly used for solubility and turbidity analysis.
The most common detection modes are absorbance and fluorescence, followed by luminescence. Researchers generally can opt for either single-mode instruments (e.g., absorbance-only) or multimode (e.g., absorbance, fluorescence, and luminescence) readers. Most companies sell a range of instruments, but some cover specific applications; Biochrom Ltd. (Cambridge, U.K.) and Bio-Rad Laboratories (Hercules, CA), for instance, focus exclusively on absorbance.
This article reviews some of the key variables to consider for those in the market for a microplate reader.
Applications of microplate readers
The first consideration in choosing a microplate reader is: What is it needed for? If you will be doing ELISAs, which are used to quantify specific proteins in a solution, you need a reader capable of making absorbance measurements in the visible range. The same is true for many protein quantitation assays, such as the Bradford assay. For nucleic acid and protein quantitation, you should choose an instrument that can read in the ultraviolet. (An absorbance mode reader is essentially a microplate-capable spectrophotometer.)
On the other hand, if you will be transfecting cells with a luciferase reporter construct and need to quantify expression, you will need a luminescence reader. If you need a fluorescence mode reader, will you be measuring simple fluorescence intensity, or do you need to run time-resolved fluorescence measurements, which are used to increase signal-to-noise by allowing background signals to decay prior to detection? Some readers can also handle specific third-party applications, such as the homogeneous time-resolved fluorescence (HTRF) assay from CisBio (Bedford, MA), or AlphaScreen® and AlphaLISA® assays from PerkinElmer (Shelton, CT). Other instruments, like PerkinElmer’s EnSpire® multimode plate reader, can handle label-free detection (for instance, using Epic® technology from Corning [Tewksbury, MA]).
If you are interested in cell-based assays, make sure your reader can specifically make such measurements from beneath the plate; because cells are typically found at the base of the well, bottom reading will improve sensitivity and signal-to-noise. There is a trend away from “mix and measure” biochemical measurements and toward more biologically representative cell-based assays. Currently, confocal-based high-content imaging systems can be used, but these can be expensive and slow, whereas microplate readers can accelerate cell-based readings and analysis.
Those who are not sure what their future needs are, or anticipate needing multiple reading modalities—for instance, in a core facility or a large lab with a variety of ongoing projects—might consider a multimode instrument, which provides greater flexibility but at a higher price.
Plate format
Most instruments can handle 96- and 384-well formats, but higher densities may require higher-end models. Higher-end models designed for high-throughput screening typically provide 1536 wells. Even fewer readers can handle the ultrahigh-density 3456-well plate format. Also, some readers cannot handle lower densities either, such as 6- or 24-well microplates for cell culture assays.
Speed and throughput
If your lab needs to run sample analysis for dozens of plates per day, time per plate can become limiting. Depending on the model, some absorbance readers may have a single reading head, while others have up to eight heads. Single-channel instruments may not all read at the same speed either. There are available systems that can read a 96-well plate in 25 sec, or 15 sec, depending on whether the head is a fixed reading or moving head. Even faster are eight-channel instruments, which can read a full plate at a single wavelength in just 4 sec.
If you are running dozens of plates per day or more, you might also consider investing in an instrument that has a plate stacker, or one that can be integrated into automated work flows. If you have to read assays quickly because the signal rapidly decays, look for an instrument that has on-board fluidics or injection capabilities.
Detectors
Also influencing reader speed is the number of detectors available for a given modality. Multiple detectors enable reading multiple wells simultaneously, or reading multiple signals from a single well. Having several photomultiplier tubes for fluorescence detection systems improves speed, and in some cases can cut the speed analysis in half.
Optics
Consider also a system’s optics. Most microplate readers are either filter-based or use a monochromator. Filters are generally more sensitive than monochromators, but only measure at specific wavelengths (thus limiting flexibility). A monochromator, which physically moves a diffraction grating to select specific wavelengths of light, provides greater flexibility than a filter, but is also slower (as each wavelength requires the grating to move) and generally less sensitive. Monochromators do provide the ability for spectral scanning, however, something that cannot be accomplished using a filter-based system.
There are alternatives to these two approaches, however. Some product advancements combine the sensitivity of a filter-based design and wavelength selection of a monochromator, such as the TUNE detection cartridge, an add-on to the SpectraMax Paradigm multimode imager from Molecular Devices (Sunnyvale, CA). Additionally, unlike monochromators, there are instruments that can measure wavelengths from 200 to 1000 nm simultaneously by passing light through a prism and projecting it onto a charge-coupled device (CCD) array, such as BMG LABTECH (Ortenberg, Germany) spectrophotometers.
Upgradability
Another variable is upgradability, or “future-proofing” your system. Few users seem to actually avail themselves of that capability, in other words, most never really upgrade, but upgradability does enable a lower cost of entry. You always will have to pay something for upgradability, but the benefit is you can have a lower entry cost if the budget does not allow for a full multimode reader at once.
Other variables to consider include temperature control, atmospheric control (i.e., the ability to control oxygen and/or carbon dioxide conditions for cell-based assays), shaking, on-board liquid handling (e.g., to enable addition of reagents prior to reading), and validation and compliance for IQ, OQ, and PQ; 21 CFR 11; GLP; and GMP. Those who might need to read exceptionally small sample volumes should look for a microplate adaptor that can handle these applications, such as the NanoQuant Plate from Tecan (Männedorf, Switzerland) and Thermo Scientific μDrop Plate from Thermo Fisher Scientific (Waltham, MA), both of which can quantify DNA or RNA in up to 16 samples using as little as 2 μL per well.
Software
Data analysis and user interface are also items to consider. Pose the following questions: Are they intuitive and easy to use, especially if you anticipate many users? Do they simplify maintenance tasks like calibration? Are there software licenses to consider?
Conclusion
Finally, consider the microplate reader as what it is: part of a work flow. Readers are fast, but it takes a lot of pipetting to process 96 or 384 wells. If your application is labor-intensive, such as ELISA, think about investing in a microplate washer. The goal is to have a microplate reader that can improve signal-to-noise and provide you with better results.
For more information on microplate readers, please visit www.labcompare.com.
Jeffrey M. Perkel, Ph.D., is a Contributing Writer for American Laboratory/Labcompare; e-mail: [email protected]. The author would like to acknowledge the following individuals for their contributions to this article: Robert Mount, Managing Director, BMG LABTECH U.K.; Kasia Proctor, Product Manager, Molecular Devices; Tristana von Will, Product Marketing Manager, Biochrom; Hanna Grano-Fabritius, Business Director, Sample Preparation and Analysis, Thermo Fisher Scientific; Michael Fejtl, Market Manager for Detection Products, Tecan Austria; and Dominic Caseñas, Product Manager, Protein Function Division, Bio-Rad Laboratories.
Please check out our Microplate Reader / Microtiter Plate Reader section for more information or to find manufacturers that sell these products