Essential components of microplate workflow
Although plate readers have existed for 30 years, and their basic operation has not significantly changed, new applications have spawned dramatic technologic breakthroughs that allow scientists to extract more data than ever from microplate experiments.
Microplate readers are long-lived and relatively maintenance-free. Lamps, which last approximately seven years with daily use, are the most vulnerable components. Many microplate readers use LED lamps instead of the traditional tungsten lamps. LEDs are maintenance free, have significantly longer life than conventional lamps, and use less energy.
Still, users should consider a maintenance contract that supports the reader, liquid dispenser, robotics, other hardware components, and software. Third-party support and maintenance are available, but most labs take advantage of their system creator’s expertise, which often extends to reagents and add-on components.
Tristana von Will, global product marketing manager at Harvard Bioscience (Cambridge, UK), notes the continuing tendency toward integrating readers into robotic systems. This is old news for fluorescence-based readers, but the influx of relatively low-cost liquid handling workstations makes it attractive for absorbance readers and microplate washers as well. ELISA assays, von Will says, comprise approximately 90% of applications for absorbance-based readers in this context.
Companies are no longer forced to choose between million-dollar robotic systems and forgoing automation altogether. “The lower-end systems address a market segment that previously could not afford high-quality automation,” von Will says. “At the same time, system software has become user friendly and does not require a programmer or automation specialist.”
Whereas larger organizations with core robotics facilities and dedicated staff are capable of adding a reader to an automated liquid handler, entry-level automation companies normally build readers into their ready-to-use systems.
The emergence of reliable, reproducible nanoliter dispensing has driven the adoption of 3,456-well microplates. “In the past, liquid handling wasn’t up to the task,” says Eric Matthews, Midwest sales manager at BMG Labtech (Cary, NC). Readers were also primitive at this well density. Older imaging-style readers provided a snapshot of fluorescence or luminescence that resembled a signal map of a microarray. But that approach lacks both the sensitivity demanded by today’s assays and the speeds required to perform highthroughput experiments.
Very-high-density plates use a fraction of the reagents and cells of standard-density microplates. Now that nanoliter dispensers are reliable and common, labs that adopt the 3,456-well format can save around 75% of reagents and cells compared with those using 1,536-well plates, and 95% compared with labs that use 384 wells. Higher density also reduces microplate consumption—by a factor of two from the 1,536 format and ten from 384 wells. Fewer plates mean less storage required, fewer manipulations, and more rapid results. For cell-based assays, culture time is shorter because cells need to grow for a shorter time to fill the smaller volume. Combined, these benefits result in lower overhead and operating costs.
High-density plates also create a need for faster reading, especially for two-wavelength assays like FRET (fluorescence resonance energy transfer). Early readers took six or seven minutes to read a microplate; the wait time has now been reduced to less than a minute. But for most users, speed was secondary to sensitivity. “That is why in the past, nobody wowed buyers by reading plates faster than their competitors did,” Matthews says. “But now that users are reading 3,456 wells on one plate, they don’t want to wait fifteen minutes for a read. So now speed matters once again.”
A related trend is the migration of assays for proteins and nucleic acids from spectrophotometers to microvolume microplates. Ninety-sixwell plates have a working volume of approximately 300 microliters per well; microvolume plate assays are in the 1-to-2-microliter range. For proteins and DNA, the assay consists of a direct ultraviolet measurement with no added reagents. Peak absorbance for DNA is 260 nm, while protein tops out at 280 nm. Direct UV assays provide concentration of either nucleic acid or protein, as well as protein contamination in DNA samples and vice versa.
“These assays previously ran individually, in cuvettes, inside spectrophotometers,” von Will explains. “In microvolume format you can read forty-eight samples at once and preserve precious sample.”
Given the proliferation of assays based on luminescence, fluorescence, and absorbance, and the diversity of biology workflows, lab managers increasingly specify multi-mode microplate readers capable of all three read modes. “Enhanced capability and flexibility are big advantages, and labs don’t need to purchase three instruments,” says Jeff Franz, global product leader for Integrated Solutions at Promega (Fitchburg, WI). “Futureproofing instrumentation will become more important as laboratory resources become scarce.”
Users are also interested in advanced detection modes such as FRET and BRET (bioluminescence resonance energy transfer), which are based, respectively, on fluorescence and luminescence. Scientists use FRET and BRET assays to study molecular interactions. While FRET has become widely popular—most reader-enabled assays involve fluorescence—few commercial BRET assay kits exist. Promega, which specializes in luminescence readers, provides tools for constructing BRET assays, based on the company’s NanoLuc™ luciferase assay system.
“The ability to multiplex standard assays with fluorescence and luminescence through a single reader allows scientists to extract more information than ever from a single well,” Franz says.
While pharmaceuticals and biotechnology are the main drivers for advanced assays and readers, the tools are increasingly adopted by the basic sciences. Many academic labs, according to Franz, are now operating as contract research organizations for large pharmaceutical interests. Advanced assay techniques naturally spill over from contract work to basic research.
The drive behind multi-mode readers is a desire for orthogonality— two or more complementary signals or assay modes in one experiment. One example would be addressing a single target through a fluorescence assay followed by luminescence mode or alpha. “Each technology has its pluses, drawbacks, and rate of false positives, which you can overcome by combining different technologies,” says Volker Eckelt, portfolio manager at PerkinElmer (Hamburg, Germany).
Orthogonal techniques may be employed in different wells, or in the same well provided the assays are compatible. One same-well experiment might incorporate a label-free assay, which provides the integrated cell response, and alpha, which targets a specific pathway.
This is the basis of phenotypic cell-based assays involving primary cells (versus immortalized or cancer cells). Primary cells are more difficult to culture than cancer cells are, but they provide greater fidelity to actual tissues and organisms. Plus, they take full advantage of the capabilities of multi-mode readers. “You can run all these assays on one platform instead of on multiple readers,” Eckelt says.
Cell-based assays have revolutionized how basic and industrial scientists test various stimuli—chemicals, drugs, pesticides, food ingredients, and others. Cells provide a biological context that in vitro assays (affinity, enzyme, ELISA, and others) lack. Improvements in visualizing events deep inside cells have, moreover, created demand for readers that not only acquire point signals but can image cellular events as well.
For example, BioTek’s latest reader, the Cytation™ 3, combines automated digital microscopy and conventional microplate detection. In conventional mode, the reader acquires whole-well signals. Microscopy enables visualization of intracellular events through interaction with stimuli.
“For scientists involved in cell-based assays, running simple assays that measure one molecule or event is not enough,” says Xavier Amouretti, manager for product marketing at BioTek Instruments (Winooski, VT). “Scientists are increasingly interested in obtaining multiple signals from complex systems and matrices, to obtain as much information as possible from a single experiment.”
Microwell-based microscopy sounds straightforward, but like most advances it relies on enabling technologies that keep cells viable and in the appropriate physiologic state for meaningful, reproducible assays. Among these features are control of temperature and vital gases such as oxygen and carbon dioxide, and automated plate shaking to facilitate oxygenation. “Together, these capabilities make readers cell friendly,” Amouretti says. “This is a very competitive market. You have to stay on top of what people are doing to remain competitive.”
The Cytation 3’s optical component (which was new for BioTek) and the operating software were developed in-house using proven industry components. The objectives come from Olympus and Zeiss, the filters are from Semrock, and the imaging CCD chip is a well-known Sony component used extensively worldwide in instrumentation. “We didn’t need to reinvent the wheel,” Amouretti comments, “but the product development was completely homegrown.”
In February 2014, BioTek received Thermo Fisher Scientific’s Extraordinary New Product Line award for the Cytation 3 at the Fisher Scientific North America Sales Meeting in Denver, Colorado.
“Cell-based assays have become indispensable in pharmaceuticals and biotechnology for their ability to deliver biologically relevant results,” adds Dr. Michael Fejtl, marketing manager for detection systems at Tecan (Groedig, Austria). “Investigators can collect information on drug interactions, including toxicity, very early in the discovery process. This helps companies identify optimal candidate molecules and avoid late-stage failures.”
Conventional cytotoxicity assays are based on endpoints: Cells grow in microwells and are treated, and the reader provides a result after a specified time period. But these assays provide only a single time point. “Looking more closely at drug interactions—for example, mode of action— is only possible with live kinetic assays,” Fejtl adds. In kinetic assays, readouts are taken at time points ranging from minutes to hours, depending on the time course of the effect. “This means the cellular environment must remain stable during the entire assay time,” says Fejtl.
One source of anomalous results is inadequate or overzealous manual cell washing, which (according to Fejtl) can “wash away the result” by removing cells or reversing a binding event. He therefore recommends automated plate washing as part of a plate reading system, to assure higher cell retention and viability.
Manual addition of growth media and feeds can result in similar issues related to cells receiving inadequate nutrition or growth environment.
In addition to keeping cells “happy,” readers must overcome cell growth anomalies to achieve a truly representative reading from a cell-based assay. Readers that focus solely on the middle of the well may miss inhomogeneities related to phenotypic distributions within wells or in cell growth patterns—for example, homogeneous clusters, 3D cultures, and non-homogeneous cultures such as normal and cancerous cells together.
To overcome this problem, Tecan employs what it calls “optimum read function,” which is incorporated in the company’s Infinity® plate readers.
A typical fluorescence experiment involves pulsing the sample with up to thirty flashes of light, and reading is done in the middle of the well. Optimal Read uses the same thirty excitation flashes but distributes them in a pattern within the well such that all cells are read. This differs from simple well scanning, a technique that also patterns the well but uses a full thirty flashes for each location. “Those take a very long time to read, even for one well,” Fejtl notes. Optimal Read takes approximately the same time as a conventional center-well read, but acquires the maximum amount of data and provides superior well-to-well uniformity.
Tecan is also one of the first vendors of lab automation to provide remote monitoring of cell health during assays through a network, including through handheld devices. Remote control is not an option due to security considerations, but users can at least alert colleagues through conventional means to replace a gas tank, change plates, or manually pipet a reagent.
Lab managers should consider the following when purchasing a microplate reader:
- Number of read modes. More is usually better, but real-world benefits depend on workflows, assay types, and the importance of orthogonality. Generally, multi-mode readers provide greater flexibility.
- Detector technology. Monochromator-based detection provides the ultimate in wavelength flexibility, enabling almost any light-based assay. Readers that use filters are fixed at specific wavelengths and are suitable for labs that perform a limited number of tests and do not engage in assay development. Hybrid systems combine both technologies.
- Future-proofing. Labs that anticipate changing or adding assays should consider the reader’s upgradability. Additional components should be readily available. A related factor is how the upgrade is accomplished— whether on-site, user-based upgrades are preferred to those that require a service visit or sending the instrument back to the vendor.
- Out-of-the-box functionality. Consider readers that users can plug into automated microplate workflows without scheduling a service visit. On-site deployments reduce downtime and build confidence.
- Interoperability. How well does the reader fit into a workflow comprising of components for multiple vendors? Interoperability with a stacker or other simple robotics is a big plus, as it reduces user contact time.
- Software. Microplate readers take hundreds or thousands of measurements—too many for the average PhD, much less a technician-level user. Data acquisition and analysis are therefore the heart and soul of microplate workflows. How easy is the software to use and adjust to different assays? Does it feature built-in protocols? How about data analysis and export format? Regulated industries should also consider data- and method-validation tools built into the software.
- Training. If training is required, will the vendor provide it at reasonable cost? Beware readers with arcane software or physical characteristics that require users to change their perspective or how they work.
- Options. Gas control, barcode scanning, shaking, and injecting increase assay flexibility for labs that require these features.
- Readers are arguably the one constant in microplate workflows. “Research labs still manipulate and process plates manually,” says BioTek’s Xavier Amouretti. “But your plate reader will be automated. Reading is the only step you can’t do manually.”
The Experts Chime In
Tristana von Will advises purchasers to think hard about potential future needs. “They’re probably conducting assays in spectrophotometers, which at some point might be converted to microplate format for the usual benefits,” she says. “Many assays traditionally run in cuvettes are quite easy to transfer into microplate format.” Reagent companies gladly provide information on the conversion. \
Along with future assay needs, von Will suggests considering wavelength flexibility. Most microplate readers use filters to acquire discrete wavelengths for specific assays—for example, protein quantification or ELISA. Advanced systems use a monochromator that allows dialing in any wavelength. Monochromator-based systems will work with any assay within the lamp’s and reader’s ability to generate and detect. “Monochromators are more versatile and appropriate for labs that develop their own assays,” von Will says.
Another consideration is data analysis software. ELISA readouts, especially at high density, can be too complex for the typical technician to upload into a spreadsheet and calculate regressions. Instruments from Harvard, for example, come with data software that automatically retrieves readings and performs calculations.
Instrument purchasers often focus on specifications— for example, dynamic range. That may be fine for some instruments, but it is not a suitable criterion for any device involved in microplate workflows. Promega’s Jeff Franz advises potential buyers to put instrumentation through its paces before buying. “Test it in an assay similar to what you run in your lab, with the same biological components,” he suggests.
A related consideration involves assays. Labs should ascertain that their proposed microplate reader purchase is compatible with assays from their preferred vendors. This is not an issue when both products are sourced from the same vendor—for example, Promega. Labs gain when a single vendor supports experimental assay designs, reagent kits, and instrumentation performance.
Franz suggests purchasing a long-term service contract. This may or may not include installation qualification or operation qualification for labs operating in regulated industries. Service contracts assure adherence to industry standards for regular maintenance, result in minimal downtime, and allow lab managers to predict more accurately the costs of maintenance and repairs.
The emergence of entry-level liquid handling systems has increased demand for must-have components such as plate readers and for other add-ons such as plate washers and shakers. “Labs we never would have expected to be open to automation are now interested in integrating a microplate reader with a liquid handler,” Franz explains. “Labs want their liquid handler and plate readers to talk to each other, so workers don’t need to redo plate maps or re-enter data.”
With integration so vital to creating automated workflows, many component manufacturers participate in SiLA (Standardization in Lab Automation), an organization that promotes instrument interoperability. Promega has recently introduced a multi-mode reader that is SiLA-compatible and that operates with other vendors’ stackers, liquid handlers, and robotics.