At the Speed of Light

High-Throughput Fluorescence Products High-Throughput Fluorescence Products (continued) PE Biosystems' FMAT 8100 HTS system Efforts by biopharmaceutical and academic laboratories to screen very large synthetic or natural compound libraries have inspired development of new and diverse forms of fluorescence technology suitable for ultrafast quantification, or high-throughput screening (HTS). This article will review the assays and instruments behind this high-speed chase. But before jumping in

Jul 10, 2000
Jorge Cortese

High-Throughput Fluorescence Products

High-Throughput Fluorescence Products (continued)


PE Biosystems' FMAT 8100 HTS system
Efforts by biopharmaceutical and academic laboratories to screen very large synthetic or natural compound libraries have inspired development of new and diverse forms of fluorescence technology suitable for ultrafast quantification, or high-throughput screening (HTS). This article will review the assays and instruments behind this high-speed chase. But before jumping into the fast waters of high-throughput (HT) fluorescence measurements (over 1,000 samples per day), the predominant techniques will be introduced.

When light hits a fluorophore molecule, it is absorbed and emitted with less energy to produce fluorescence. The emission intensity is a measure of fluorophore concentration. Alternatively, light absorption by a fluorophore can occur close to an appropriate acceptor that receives it and emits a photon (fluorescence resonance energy transfer or FRET). Proximity between fluorophores measured by FRET precisely reflects binding of soluble and membrane-bound molecules.1

Time-resolved fluorescence (TRF) is a technique that improves fluorescence quantitation. TRF uses rare-earth lanthanides to provide a long-lived fluorescent signal. Measuring this type of fluorescence after a time delay eliminates interference from short-lived background fluorescence (50 msec or less), improving sensitivity.2 Wallac(tm) DELFIA(r) (Dissociation-Enhanced Lanthanide FluoroImmunoAssay) technology from PerkinElmer Life Sciences of Gaithersburg, Md., is a form of heterogeneous TRF that relies on nonfluorescent chelates that can be converted to highly fluorescent forms using a developing solution. Analogous assays based on FRET combined with TRF include Wallac LANCE(tm) technology, the homogeneous chelate chemistry from PerkinElmer Life Sciences, and HTRF(r) (Homogeneous Time-Resolved Fluorescence) from Packard Instrument Co. of Meriden, Conn.

A host of assays are based on fluorescence polarization (FP). Immobile fluorescent molecules illuminated with polarized light emit photons with similar polarization; depolarization is proportional to molecular rotation. This allows discrimination between bound and free states, because the binding of a fluorescently labeled molecule to a target restricts its motion (higher FP). The [FP]2(tm) solution phase assays by NEN(r) Life Science Products Inc. of Boston use Fluo-peptides(tm) (BODIPY(r) TMR-labeled, biologically active peptides) as FP-detectable ligands for many G protein-coupled receptors (GPCRs). Another example is the CoreHTS(tm) line of FP assays from PanVera Corp. of Madison, Wis. CoreHTS assays are available for kinases such as protein kinase C. They use FP to measure competition for binding to antiphosphotyrosine antibodies between synthetic fluorophosphopeptides and the nonfluorescent phosphopeptides generated by kinases. PanVera's HTS-binding assays for estrogen receptors (ERs) employ Fluormone(tm) (ES2), a fluorescein-labeled estrogen ligand. In the absence of competing ligands, the ER/ES2 complex has high FP; high-affinity ligands decrease FP.

HT Microplate Readers

A number of microplate readers that allow robotic handling of microplates (20-plate stackers are standard) aid the transition from single sample to HT microplate assay work. For example, the MFX(tm) microplate fluorometer from DYNEX Technologies Inc. of Chantilly, Va., is compatible with the Twister(tm) Universal Microplate Handler from Zymark of Hopkinton, Mass.

The widely used Fluoroskan instruments from Labsystems of Franklin, Mass., are robot friendly. The Fluoroskan Ascent FL is both a fluorometer and a luminometer, remotely controlled by Ascent software that sets up predefined assays. Up to three liquid dispensers can be used, and the Assist(tm) plate-handling device provides a versatile plate stacker. A simplified instrument for cell-based work (for example, gene reporter assays), the Fluoroskan Ascent CF can read plates from below with sensitive fiberless optics. Also with the HTS cell biologist in mind, PerkinElmer Life Sciences offers the FLite(tm) fluorometer that can scan the entire area of each well or take readings from any user-defined point in the well. The instrument can also assess different labels sequentially from the top (open lids) or bottom (closed lids) of microplates with up to 864 wells.

For multiple optical measurements, Bio-Tek Instruments of Winooski, Vt., offers the FL600, a fluorescence microplate reader that also reads luminescence (glow) and absorbance assays. The instrument reads microplates with six to 384 wells and features filter wheels for up to six filters, a five-decade fluorescence range, and the ability to read fluorescence from the top or bottom of the plate, with automatic software switching. The FL600 has temperature control to 50°C for temperature- sensitive assays. It reads plates in less than 30 seconds and carries out complete ELISA analysis and endpoint and kinetic measurements. It includes high-end options such as well area screening, validation, and cutoff calculations and comes with KC4(tm) data reduction software for Windows(r).

TECAN U.S. of Durham, N.C., offers two robot-compatible instruments, the SPECTRAFluor and SPECTRAFluor Plus, which measure fluorescence and absorbance. Both instruments use pulsed excitation light generated by a xenon flash lamp, include top/bottom reading capability, and are available with data reduction and reader control software. The SPECTRAFluor Plus offers a deeper UV range (230-700 nm), increased sensitivity, and a glow-type luminescence measurement mode. Both instruments can accommodate 384-well plates; however, the SPECTRAFluor Plus can perform fluorescence readings in 1,536-well microplates and luminescence in a 96-well format.

The LS Reader(tm) from PerkinElmer Instruments of Wellesley, Mass., is a dedicated fluorescence plate reader with kinetics, matrix scanning (circular and square formats), dual ratio measurement, and top and/or bottom reading capabilities. The LS Reader accommodates a variety of formats including 1,536-well plates, filters, petri dishes, or Terasaki plates. A state-of-the-art photon counting system provides high sensitivity, and adjustable, computer- controlled source intensity allows reproducibility of analytical conditions.

Multifaceted Fluorescence

The complex needs of HTS have encouraged development of instruments for multiple fluorescence methods. A popular addition to standard fluorescence measurements is TRF capability. Molecular Devices of Sunnyvale, Calif., exemplifies this approach with the SPECTRAmax(r) GEMINI XS, a novel dual-monochromator microplate spectrofluorometer that eliminates the need for filters to choose excitation and emission wavelengths. It can read a single region in the well center or scan multiple points for fluorescence, TRF, or luminescence. The GEMINI XS communicates with robotic/LIMS systems through SOFTmax(r) PRO 3.1 software, enabling validation and GLP/GMP compliance.

Packard Instrument Co.'s Discovery(r) AD HTRF microplate analyzer is designed for HTRF assay development and measures 20 96/384-well microplates. The Discovery HTS model increases throughput to 50,000 samples per day with its 40 plate stackers. The latest addition to Packard's line of microplate readers is the Fusion(tm) universal microplate analyzer. The Fusion analyzer is designed to measure TRF, HTRF, FRET, top and bottom fluorescence intensity, absorbance, and luminescence with a single instrument. It is capable of reading microplates from six to 1,536 wells to support basic research, assay development, and HTS. It can be configured with 40 external plate stackers for unattended reading of 61,440 samples in approximately four hours.

A widely used fluorescence microplate reader by PE Biosystems of Foster City, Calif., is the CytoFluor(r) 4000 Fluorescence Multi-Well Plate Reader. This system provides easy-to-use software, six excitation and emission filter positions, a 5 log dynamic range, a lower detection limit of 8 fmol of sodium fluorescein per well, and an optional automated plate handler for up to 80 microplates (six to 384 well). The temperature-controlled model features temperature uniformity of +/- 0.3°C.

Since measuring FP requires a different instrument design, some machines add this feature in a separate fluorometer. For example, the FLUOstar Galaxy from BMG Labtechnologies GmbH of Offenburg, Germany, measures fluorescence, TRF, flash and glow luminescence, and absorbance; the POLARstar Galaxy model adds FP capability. Up to eight different filters can be used, allowing all five measurements in 96/384-well microplates. Fluorescence and TRF measurements can be done in 1,536-well microplates. Other complementary instrument systems are the 1420 Wallac VICTOR2(tm) multitask, multi-label counter (fluorescence, TRF, luminescence) from PerkinElmer Life Sciences and the VICTOR2V model, which adds FP, absorbance, and preprogramming with common assay protocols (DELFIA and LANCE). The VICTOR models can process microplates with up to 1,536 wells, supporting fast kinetics and dual-ratio measurements. A robot-compatible machine with a high degree of precision in FP or standard fluorescence modes is the POLARION from TECAN. The instrument uses fast, motorized operation of the polarizers to read 96-well microplates in less than 100 seconds. The POLARION is also capable of reading in the 384-well format and, like all TECAN instruments, is available with data reduction and reader control software.

Moving to higher HTS-FP performance levels, the Criterion(tm) line of instruments from LJL BioSystems Inc. of Sunnyvale, Calif., carries out fluorescence, luminescence, TRF, HEFP(tm) (high-efficiency FP), and absorbance detection. The detection performance is achieved by SmartOptics(tm), a unique hardware system exploiting flexible configurations of light sources, filters, detectors, and a novel three-axis positioning. The optic system also features progressive z-axis focus variation that collects maximal light, avoiding contaminating signals from the well walls and bottoms. The Criterion instruments support dual-mode assays or two-wavelength assays such as TR-FRET. Within LJL's Criterion product line, the Analyst(tm) AD is a cost-effective solution for assay development, while the Analyst HT reaches the HTS range, reading 200 384-well microplates in less than nine hours. In addition, the Analyst HT's optical and detection settings are automatic, allowing integration into robotic systems. LJL has recently introduced Screen Station(tm), an integrated solution for liquid handling of 1,536-well format microplates and reading of microplates using most single- or dual- fluorescence modes. Also very fast, the ULTRA reader from TECAN enables 1,536-well measurements of fluorescence (UV and visible), glow luminescence, FP, TRF, and absorbance. A 1,536-well microplate can be read in less than one minute. To accommodate commercially available chromophores, the ULTRA utilizes multiple pairs of excitation and emission filters as well as multipositional probe-specific dichroic mirrors.

Other systems image the entire microplate rather than measuring each well. The Wallac 1442 Arthur(tm) multiwavelength fluoroimager from PerkinElmer Life Sciences combines three sources of laser illumination and a horizontal- scanning CCD camera to allow measurements from up to four fluorophores and includes luminescence and absorbance measurement capability. Wavelengths are freely selectable from the UV to near-IR range, and the instrument is compatible with 1-D or 2-D gels and membrane or microscope slide arrays. Also from PerkinElmer Life Sciences, the Berthold NightOWL(tm) fluoroluminometer explores ultrasensitive imaging of low-level fluorescence and luminescence applications. Although primarily for microscopic imaging, it detects steady-state or kinetic processes in 96-well microplates.

The Fluorometric Imaging Plate Reader I (FLIPR(r) I or "Flipper" for friends) by Molecular Devices is a remarkable instrument for homogeneous kinetic and cell-based fluorescent assays aiming for HT. It can simultaneously deliver compounds to wells and gather images in 96 channels; its optical system can discriminate cell monolayers from bulk solution. The upscale FLIPR384 can obtain impressive real-time kinetic information in 384-well microplates, generating 60,000 traces per day. The FLIPR has been used to measure intracellular pH or calcium and plasma membrane electric potential.

Confocal technology can also be used for fluorescence quantitation. PE Biosystems, in collaboration with Becton Dickinson and Co. of Franklin Lakes, N.J., has developed the FMAT(tm) 8100 HTS System, a fluorescence-based, macroconfocal imaging system for direct quantification of cellular or bead-based fluorescence, covering a broad application portfolio. The FMAT system enables detection in the red region of the spectrum, minimizing background fluorescence interference from cells, plastics, and library compounds; a 96-well microplate can be read in seven minutes. It is interfaced with Zymark's Twister robot. Many applications have been implemented for the FMAT system, including homogenous fluorescent-linked immunosorbent assay (FLISA), cell-surface receptor binding assays, and cell viability and apoptosis-related toxicological screening.

Optical quality and robotics for imaging high densities of small wells are reaching their limits. To overcome these hurdles, Carl Zeiss of Jena, Germany, has developed a multimode reader that uses an array of 96 miniature lenses. The 96 light rays are mapped on a CCD sensor surface for fluorescence and luminescence measurements; for absorbance, a special array of photodiodes is used. Genuine kinetic experiments can be conducted simultaneously for all 96 wells (a well is read in 20 seconds) or in a few steps for 384/1,536-well microplates.

The Assay Corner

A number of companies offer fluorescence- based assays that can be adapted for HT. In particular, Molecular Probes of Eugene, Ore., has developed numerous fluorogenic assays that are HT compatible, such as its line of EnzChek(r) protease assays. PanVera Corp. produces HTS-FP assays (CoreHTS) that measure enzymatic activity or ligand binding precisely enough to characterize IC50 values. Fluorescence-based assays are also available from CLONTECH of Palo Alto, Calif., and Pierce Chemical Co. of Rockford, Ill.

Optimizing assays is never easy, and some choose to turn development and/or implementation over to the professionals. Aurora Biosciences Corp. of San Diego, a big-league player in this market segment, provides contract-based access to fluorescence-based screening--including target validation--all the way to ultra-HTS. Aurora has a range of fluorescent reagents and sophisticated platforms, including unique voltage-sensitive probes and the VIPR(tm) reader for ion channel HTS. The company's repertoire includes systems for measuring receptor binding (using FRET-competent dual-labeled peptides), proprietary green fluorescent protein (GFP) variants, and ß-lactamase reporter activity. Another high-volume service provider is Discovery Partners Ltd. of Allschwil, Switzerland, a subsidiary of Discovery Partners International of San Diego that offers the HTS Factory(tm) contract system for development of assays based on fluorescence, homogenous TRF, FP, or luminescence in addition to large chemical libraries for customized discovery services. PanVera and LJL Biosystems' ADEPT(tm) team also have HT assay and reagent development services.


Molecular Devices' FLIPR I System

Drug Racers

Pharmaceutical companies screening very large libraries have pushed HTS to the ultra-HTS level, beyond or around 100,000 assays per day. Examples are the ultra-HTS (UHTSS(tm)) platform from Aurora Biosciences and the ultra-HTS workstations jointly developed by Carl Zeiss and Hoffmann-La Roche of Nutley, N.J. The latter platform uses core technology such as high-performance, multimode readers with a modular ensemble of robotic stations. Another platform that provides this level of throughput is the new LEADseeker Homogeneous Imaging System from Amersham Pharmacia Biotech of Uppsala, Sweden. The homogeneous ultra-HTS system is based on imaging technology, but offers fluorometric, luminescent, and radiometric options as well.

EVOTEC BioSystems AG of Hamburg, Germany, also develops custom assays and offers HTS services, as well as technology access and transfer agreements for ultra-HTS. EVOTEC combines detection technologies--some unique to the company--such as fluorescence, depolarization, fluorescence lifetime, FRET, fluorescence quenching, and fluorescence correlation spectroscopy (FCS). EVOTEC's ultra-HT EVOscreen(r) platform converts traditional microplates to Nanocarriers(tm) (high-density plates with more than 2,000 wells) using Micro-To-Nano (M2N) robotics and allows in excess of 100,000 assays to be performed per day. The technology offers applications to gene-to-screen strategies (PICKOscreen) and functional ultra-high performance screening (HPS) assays for a wide variety of targets (for example, orphan GPCRs and enzymes). Solutions for data storage and database building are integrated into the platform.

Some ultra-HTS instrumentation even allows screening to be carried out in-house. A member of LJL's Criterion line of instruments jumps into ultra-HT territory. The Acquest(tm) microplate fluorometer has the capacity to analyze 1,536-well microplates with assay volumes as low as 2 µl. The instrument has multiple detection modes (fluorescence, TRF, FP, luminescence, and absorbance), a wide dynamic range (8 log), and calibration-free optics. Reading over 200,000 wells per day with up to 1,000 times the sensitivity of imaging systems, this is a serious contender for ultra-HTS work and HTS robotic system integration. A new ultra-HTS microplate imager, the Wallac ViewLux(tm) from PerkinElmer Life Sciences, allows measurement of microplates with up to 1,536 wells in fluorescence, TRF or TR-FRET, FP, HEFP, luminescence, and absorbance modes. The TRF capability affords labs using or switching to DELFIA or LANCE assays an easy transition to ultra-HTS. The system is very fast, reading a plate in less than 30 seconds, with a throughput of more than 200,000 per hour. Detection uses a Peltier-cooled CCD camera coupled to an optimized telecentric lens; for automatic operation, the ViewLux supports robotic loading, batch-mode operation, and four barcode readers.

The Future, Now

HT fluorescence is growing in multiple directions to cover such biochemical and cellular characterization as proteomics research and evaluation of genomic targets. Drug-protein interactions can be studied using fluorescence assays, and 3-Dimensional Pharmaceuticals Inc. of Exton, Pa., has developed Thermofluor(r), a miniaturized process that measures ligand binding to proteins by differentially analyzing thermal unfolding curves. Because Thermofluor measures a physical property common to all proteins, assay development time for new targets is very short.

The impending tsunami of genomics-proteomics data has led Luminex Corp. of Austin, Texas, to develop an assay universe in the form of Suspension Arrays(tm). The technology uses proprietary, precision ratio labeling with two fluorophores to create 100 distinguishable microsphere types with distinct "addresses." Multiple fluorescence-based binding assays, each corresponding to an address, can be carried out in the same tube.3 Known as Laboratory Multiple Analyte Profiling (LabMAP(tm)) technology, this system operates as a parallel processing, high-speed analysis unit (Luminex 100(tm) instrument) with an X-Y Platform (XYP(tm)) robotic component. It will expand not just HTS work but also diagnostics. In the future, up to a million simultaneous reactions will be studied at once.

FCS is another innovative assay technology being adapted for HTS. FCS has been around for a while, but recent upgrades in speed and sensitivity have allowed it to reach ultra-HT levels.4 Fluctuations of light emitted by a fluorescent molecule look like experimental noise but contain information about the number of fluorescent particles (concentration) and their relative size (diffusion rate). To unravel this data, light changes--peaks and valleys--are "correlated" over time; hence the name FCS. Using confocal technology to observe femtoliter-size volumes, FCS is the most sensitive (10-15-10-18 M) fluorescence technique. Researchers are using FCS to look deeply into receptor-ligand or nucleic acid interactions and to substitute some PCR-based techniques with FCS' single-molecule detection. All of this will soon be possible, and Carl Zeiss has launched a commercial FCS instrument, the ConfoCor 2, which can be purchased as a stand-alone FCS system or combined with a LSM 510 confocal microscope.

The upcoming frontier in HT fluorescence is application to cell biology. D. Lansing Taylor, president and CEO of Cellomics(r) Inc. of Pittsburgh, Pa., stresses that "the limitations of the information gathered by genomics and proteomics are being overcome by the new tools of cellomics; this is leading to the industrialization of cell biology." Studying the information contained in genes and their coded proteins alone will not elucidate complex biochemical pathways. Cell-based HTS assays that look deeply into cell physiology will be required. Cellomics calls this "High Content Screening" (HCS); in HCS, cellular protein localization and function are studied with fluorescently labeled molecules or autofluorescent proteins, such as GFP, using fluorescence, TRF, FRET, FP, fluorescence lifetimes, and FCS.5

For HCS, Cellomics has developed the ArrayScan(r) II, an autofocusing instrument that registers spatial and temporal events in cells. The ArrayScan II system uses a white light source, providing multiple fluorescence channels. A high-capacity plate stacker accommodates standard microplate formats. To be released later in the year is the ArrayScan Kinetics Workstation, boasting higher HCS throughput, FP capability, automated plate handling and fluidics, and eight fluorescence channels. Cellomics also offers integrated, layered software: Cellomics Store, a terabyte-size database, supports data from microplate readers or imagers such as the ArrayScan and Molecular Devices' FLIPR; Cellomics DataViewer has drill-down capabilities to give extensive data at the plate, well, or cell level; Cellomics Screen enables management and scheduling of screening runs and analysis of dose-response curves and cross-correlating parameters; and Cellomics Discover, the highest-level bioinformatics platform, includes data mining technology and pattern recognition. Cellomics has also teamed up with Carl Zeiss and Molecular Probes to take HCS to the ultra-HTS level.

Following this trend of integrated cell-HTS solutions, Universal Imaging Corp. of West Chester, Pa., is introducing a Cell Based Screening System. This system fully automates common microscopy for cell-based assays in multiwell plates for cell proliferation, cell viability with live dead analysis, protein expression and redistribution, or colocalization using multiple probes. The fully automated optomechanical platform for the system was designed in collaboration with Sutter Instruments of Novato, Calif., and Nikon U.S.A. of Melville, N.Y. Four filter wheels control illumination, high-speed focusing, and integration with several currently available robotic plate handlers. The end result is eight measurable probes per well in 48-, 96-, 384-, and 1,536-well microplates, motorized control of objective magnification, and an intuitive Image Viewer/Analyzer software package for automating image analysis using the company's MetaMorph software tools. The system is distributed and supported by Becton Dickinson.

If the reader has made it this far, the conclusion will be clear: High-throughput fluorescence technology is pervading the continuum between genetic, functional, and cellular information and may soon be the predominant form of molecular characterization in both academia and industry. ... If only scientists could make light go faster. S

Jorge Cortese (jorge_cortese@mindspring.com) is a freelance writer in Durham, N.C.

References

1. J.D. Cortese, C.R. Hackenbrock, "Motional dynamics of functional cytochrome c delivered by low pH fusion into the intermembrane space of intact mitochondria," Biochimica et Biophysica Acta, 1142:194-202, 1993.

2. I. Hemmilä, S. Webb, "Time-resolved fluorometry: an overview of the labels and core technologies for drug screening applications," Drug Discovery Today, 2:373-81, 1997.

3. J.D. Cortese, "Sphere of influence," The Scientist, 14[6]:16, March 20, 2000.

4. T. Winkler et al., "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proceedings of the National Academy of Sciences, 96:1375-8, Feb. 8, 1999.

5. K.A. Giuliano, D.L. Taylor, "Fluorescent-protein biosensors: new tools for drug discovery," Trends in Biotechnology, 16:135-40, 1998.