Microplate readers have become lab benchtop standards, a boon for researchers who require the instruments to quan-a tify chemical reactions using a range of light-detection methods. Their ability to screen entire multiwell plates in seconds affords high-throughput, parallel processing of small sample volumes. But not all microplate readers are equal, and the need for more sensitive instruments has split the market between absorbance readers and those capable of multiple-detection modes, including fluorescence and luminescence measurements.
Whereas absorbance microplate readers use visible or ultraviolet light for detection, fluorescence readers bombard samples with light that is then absorbed by fluorophores and emitted at longer wavelengths. The method, often used in cell-based studies, can offer much higher sensitivity than conventional UV-Vis absorbance detection. In addition to standard fluorescence detection, some microplate readers are designed to scan for time-resolved fluorescence and fluorescence polarization.
Luminescence readers, in contrast, detect without excitation light, relying instead on...
A MARKET EVOLVES
According to a 2002 report of the US microplate reader market by San Carlos, Calif.-based PhorTech, fluorescence, luminescence, and multimode models accounted for about half the microplate readers purchased since 1999. Researchers sought out more multimode readers than dedicated fluorescence and luminescence tools because of their added flexibility, the report stated.
Boston-based PerkinElmer Life and Analytical Sciences dominated the multimode reader market with 52.8 percent of the marketplace, according to the report, followed by longtime industry heavyweight Molecular Devices of Sunnyvale, Calif., and Switzerland-based Tecan. Bio-Tek Instruments of Winooski, Vt., and BMG Labtech of Offenburg, Germany, rounded out the market's top five manufacturers.
With such a range to choose from, researchers searching for new luminescence or fluorescence microplate readers should consider how much flexibility they need in a potential instrument, as well as their intended applications. Throughput requirements should also be considered, as many readers are built to handle multiple microplate formats (96, 384, and 1536 wells).
FLEXIBILITY VS. DEDICATION
Multimode readers for luminescence and fluorescence are designed for researchers who require more than a single detection mode. Simple multimode instruments add either fluorescence or luminescence detections to the standard absorbance function. Higher-end models include added fluorescence capabilities such as fluorescence polarization or time-resolved fluorescence (TRF), and fast kinetic fluorescence as well as flash- and glow-luminescence functions.
For example, the new PHERAstar multimode reader from BMG Labtech automatically adjusts filter and optics settings to provide a range of fluorescence, luminescence, and absorbance detections. The setup allows users to optimize the reader for the specifics of each run.
PerkinElmer's EnVision multimode microplate readers can be optimized with the company's bead-based AlphaScreen chemistry to run Amplified Luminescent Proximity Homogenous Assays (ALPHA), which generate fluorescence and luminescence signals from binding interactions between donor and acceptor beads. The combination offers femtomolar sensitivity and limits background interference caused by fluorescence signals from microplates, assay components, or samples. Targeted for drug discovery, EnVision systems are available in medium- and high-throughput configurations. Both models can screen 5 μl per sample and can detect by absorbance, luminescence, fluorescence, fluorescence polarization, and TRF.
Dedicated readers have their place, too, both in small academic labs needing simple devices and in high-throughput labs, in which fluorescence and luminescence measurements are conducted often enough that a single multimode instrument could cause a bottleneck. Dedicated readers can also be tailored for specific end-user applications, such as the live-cell and bead studies targeted by the fluorescence-based 8200 Cellular Detection System offered by Applied Biosystems of Foster City, Calif.
TO INJECT OR NOT INJECT
Most fluorescence and luminescence microplate instruments automatically handle standard plate runs. Simpler readers are typically tailored for glow luminescence or fluorescence experiments, which can provide illumination for more than an hour.
Flash luminescence and fast kinetic fluorescence studies require accurate detections made just seconds after a reagent is added to a well, when the reaction generates its strongest signal in a flash of light. Higher-end instruments include injectors or dispensers to add reagent to a well in time for its detector to record the reaction.
The Luminoskan Ascent luminometers available from Thermo Electron of Vantaa, Finland, for example, can be fitted with three onboard injectors that can dispense reagent across a volume range of 5–1,000 μl in 1 μl increments.
Some manufacturers offer adjustable injectors, such as those on the Lmax II and Lmax II 384 luminometers from Molecular Devices. Users can reverse the direction of flow through the injectors to recover expensive reagents from the dead volume in the fluidics. Lmax jet injector technology also speeds mixing to boost data quality.
PLATE FORMAT AND THROUGHPUT
Because most microplate footprints are standardized (128 mm × 86 mm), most readers can handle multiple formats, including plates with 6, 96, 384, and 1,536 wells.
To maximize throughput, instrument manufacturers have equipped their readers to be robot-ready, allowing the use of multiplate stackers and liquid handlers to boost productivity. Beckman Coulter of Fullerton, Calif., has new DTK 800 and 880 readers, which are benchtop multimode instruments that can either stand alone or work with Beckman's Biomek automation platforms for higher throughput.
The multimode readers offered by Carl Zeiss MicroImaging of Thornwood, NY, push throughput even more. Based on the plate::vision multimode reader from parent company Carl Zeiss Jena of Germany, the plate::screen reader scans 50,000 wells per hour, taking one second for a 96-well plate and 20 seconds for a 1,536-well plate.
Though the choices can seem overwhelming, one thing is certain: With the range of reader options expanding at a rapid pace, users can find an instrument to suit virtually any need.
Tariq J. Malik