Luminometers and Fluorometers

Date: November 24, 1997 Chart To most people, a luminometer is something that probably measures "luminos," while a fluorometer measures "fluoros," As simple as that sounds, these analyzers have been occupying valuable bench space in laboratories for decades. Though maybe not considered new and hot techniques in the biotechnology arena, luminometry and fluorometry continue to provide scientists with reliable answers to molecular questions. LabConsumer set out to explore options available to scientists involved in luminometry and fluorometry. As with other popular biotechnology instrumentation, manufacturers in these two fields appear to be focusing on expanding sample formats and increasing throughput, which should definitely be considered in the purchasing decision. Manufacturers are also developing specialized devices with temperature control, automation options, and various software programs for specific applications. Luminometers and fluorometers are discussed briefly, and a comparative table of luminometers, dispensing luminometers, and fluorometers is provided. The measurement of chemiluminescence (light emitted from a chemical reaction) and bioluminescence (naturally occurring luminescence that is often enzyme-catalyzed) is made possible by the use of luminometers. The majority of these devices utilize photomultiplier tubes that detect light emitted from the reaction, with the light reaching the tube being proportional to the concentration of the limiting reagent in the reaction. Luminometry provides a higher signal-to-noise ratio than fluorometry. Common applications of luminometry include reporter gene assays-used to study gene expression and regulation-and ATP determinations. The Lumi-ScintTM from Bioscan is a small, benchtop luminometer optimized for counting luminescence and liquid scintillation counting (LSC). Using photomultiplier tube detection, the Lumi-Scint reads one tube or LSC vial at a time. Applications include quantitative polymerase chain reaction (PCR) and reporter gene assays. Bioscan also offers the Lumi-One Luminometer, which has the same features as the Lumi-Scint except LSC capabilities. MGM Instruments provides the compact OPTOCOMPTM-P Portable Luminometer, which features 8-hour battery operation and a standard printer for paper documentation. The user is guided by the unit's menu-driven software, and data reduction is limited to raw counts to provide cost-effective luminescence assays. The Lumi-Tec from St. John Associates is available in three configurations: a test tube model for discrete samples, a flow cell model, and a flow cell model with an integral mixing tee for the addition of a reagent. The test tube instrument uses glass or polystyrene cuvettes, while the flow cell models have a 70 ml flow cell. (Other cell volumes are available on request.) Data are displayed on an LED readout. Packard TopCount® NXT For microplate luminometry, Bioscan provides the Lumi-96, which accepts 96-well microplates and is run by WindowsTM software. Analytical Luminescence Laboratory Monolioght® 3010 The LumiCountTM Microplate Luminometer from Packard Instrument Company features a compact design, making it ideally suited for integration with automated liquid handlers or robotic systems. The WindowsTM software enables measurements on 6- to 96-well micro-plates and allows programmable incubation and temperature control from ambient to 45°C (±0.1°C). The WindowsTM NT-based TopCountTM NXTTM Microplate Scintillation and Luminescence Counter measures a variety of radioisotopic and enhanced luminescence assays-2, 6, or 12 samples at a time-and can accept 24-, 96-, or 384- well microplates and microfuge tubes. The TopCount NXT, with its patented crosstalk-free measurement design, delivers high sensitivity for both scintillation assays and glow luminescence chemistries. The TopCount can process 50,000 samples per day with a 40 plate stacker. Cardinal Associates FluteTM 400 Analytical Luminescence Laboratory manufactures the programmable Monolight 3010 Luminometer. The Monolight 3010 comes with one or two variable volume automatic injectors, a built-in thermal printer, and reads one sample cuvette. The Monolight is controlled by a microprocessor, and is suitable for all bio- and chemiluminescent applications. Software permits the storage of 40 counting protocols for measuring different modes. The software also is capable of performing the inject-read-inject-read measurement, as is required for measuring dual luciferases. EG&G Wallac LB9507 The FLyteTM 400 Automated Tube Luminometer from Cardinal Associates allows reading and reporting of results of 114 samples in as little as seven minutes. In its standard configuration, the FLyteTM 400 comes with a single high- precision dispenser, but it can be customized to simultaneously deliver two different reagents into each sample tube for multiple reagent protocols. The FLyteTM400 can be further customized with the programmable Cardinal Thermal Control unit, which maintains the temperature of the sample chamber at 37°C (±1°C). Distributed in the United States by EG&G Wallac, the AutoLumat LB 953 is optimized for bioluminescence, cellular chemiluminescence, and immunoassays. The LB 953 can process a sample chain of up to 180 sample carriers, using up to three injectors simultaneously. The LB 953 also features user- interchangeable filters, optional temperature control of the sample compartment (from 5°C above ambient to 45°C), and a wide selection of software programs for various applications. The Lumat LB 9507, also from EG&G Wallac, features a motor- driven rotating chamber for two tubes-one for measuring and one for loading and unloading-allowing tubes to be loaded and unloaded during sample measurements. Up to two programmable, automatic, variable volume injectors can be used on the LB 9507. Measured raw data are simultaneously sent to a serial port, which allows data acquisition from an external computer, the software of which is dual luciferase assay compatible. MGM Instruments OPTOCOMP I The OPTOCOMPTM I and OPTOCOMPTM II from MGM Instruments are tube counting luminometers qualified for all chemiluminescent and bioluminescent reagents available. The OPTOCOMP I can accept a single test tube, while the OPTOCOMP II can accept up to 250 tubes and can be programmed to process up to 11 user-programmable protocols. Both machines are prefitted with up to two automatic, fixed-volume injectors and feature a 40- character graphic thermal printer for reporting of protocol identification, time, date, operator name, and numerous results formats. To eliminate static discharge interference with luminescence readings, BBI-Source Scientific integrated an innovative static-suppress feature in its E/LUMINATM II Luminescence Analyzer. The software of this unit is designed for assay versatility, allowing simple set-up menus for injection timing and kinetic read modes. An extensive menu of data reduction, standard curve fits, concentration units, and results flags is available for custom defining user protocols. The tracking program of the E/LUMINA II monitors reagent usage and provides reports during operation. The TD-20/20 from Turner Designs can be outfitted with programmable single or dual internal auto injectors and is capable of reading a 35 mm culture dish, a 28 mm scintillation vial, test tubes, and microfuge tubes. The TD-20/20 includes software for Promega's Dual-LuciferaseTM Reporter Assay System and has a kinetics feature for reactions with unstable light output. An optional printer is also available. EG&G Wallac MicroLumatPlus LB 96V The LUMIstar from BMG LabTechnologies is a fully automated microplate-based luminescence reader equipped with two high- precision syringe pumps. Incubation of the LUMIstar is fully programmable via PC, and measured temperature is displayed on the screen in real time. The front-loading plate transport and the communication protocol allow the LUMIstar to be linked to integrated robotic analyzers. The new MLXTM Luminometer from DYNEX Technologies is designed for glow endpoint, glow kinetic, or flash reactions and is controlled by DYNEX's WindowsTM-based software system RevelationTM. As a component of Revelation, Assay WizardTM utilizes a step-by-step tab dialogue approach to programming all operating and data reduction aspects of an assay. The MLX accommodates up to three positive displacement syringe dispensers. Revelation has a new software application that accomodates the Promega Dual LuciferaseTM Reporter Assay (DLRA). rosys anthos Lucy I EG&G Wallac's MicroLumat-Plus is a microplate luminometer with up to three variable volume injectors. The MicroLumatPlus can be used as a stand-alone instrument for sample measurement and data reduction, while the WinGlow software package offers the possibility of controlling the instrument with a WindowsTM-based system that is dual luciferase assay compatible. The MicroLumatPlus can also be outfitted with a multizone incubation module for temperature equilibration. Tropix TR717 EG&G Wallac also offers the MicroBetaTM JET, which can handle 24- or 96-well microplates, filtermats, and tubes up to 45 mm in height, and provides luminescence reading and liquid scintillation counting. One or two modules can inject up to two reagents into the reading position of six detectors, or one module can inject up to four reagents into the reading position of one detector. Lastly, EG&G Wallac is the exclusive distributer of the Hidex Oy Triathler. The Triathler is also designed for luminometry and liquid scintillation applications. This flexible unit can accept microcentrifuge tubes, mini LSC vials, and normal LSC vials by way of adapters. Its portability makes it an excellent candidate for field surveys. The anthos lucy 1 from rosys anthos is a microplate luminometer and photometer. Up to two precision dispensers are software controlled for delivery from 10 ml to well capacity in increments of 1 ml. Also, the plate transport provides a shaking function for mixing. The measurement chamber is Peltier controlled, allowing temperatures to be adjusted from 4°C below ambient to 40°C in steps of 1°C. L1Com under WindowsTM or MacL1Com for MacintoshTM-based systems enables remote operation of lucy 1 including test execution, result upload, test upload, and test download. SFRI's lumax 3 is a 96-well microplate luminometer equipped with two to four injectors, allowing both flash and glow assays to be performed. The lumax 3 is self-calibrating, and results can be obtained in as little as 40 seconds. The unit has a very small footprint, is lightweight, and has an incubation range that extends to 55°C. The lumax 2 from SFRI is about half the size of the lumax 3 and can read glow assays. SFRI also provides the Alpha 4LS, which the company claims is the only completely automated microtiter plate system that reads both optical density and luminescence simultaneously. The system accepts up to 384-well plates and is only 41 inches wide. The TR717TM Microplate Luminometer from Tropix reads 96- or 384-well plates and measures all types of chemi- and bioluminescence (flash and glow kinetics). Up to two programmable reagent injectors expand assay design capabilities. The TR717 is controlled via an internal keyboard, but its intuitive and easy-to-use Windowsª software can greatly increase data analysis and handling capabilities. Tropix also provides 12 customized kits and numerous reagents for luminescence assays. An optional robotics package allows for the integration of the TR717 into most robotic systems. Tropix also provides 12 customized kits and numerous chemiluminescent reagents. Pharmacia Biotech DyNA Quant 200 Fluorescence is the molecular absorption of light energy at one wavelength (excitation) and its subsequent re-emission at another wavelength (emission). Fluorescent compounds are characterized by unique excitation and emission spectra; no two compounds have the same fluorescence signature. This makes fluorometry a highly specific analytical method. While some molecules are inherently fluorescent, others must be coupled with other molecules to form a fluorescent compound. Fluorometry is highly sensitive (more sensitive than spectrophotometry, yet less sensitive than luminometry), simple, and generally inexpensive. A few of the numerous applications of this technique include DNA/RNA quantitation, enzyme activity, hydrocarbon detection, tracer dye studies, and chlorophyll measurement. Fluorometers generate a specified wavelength of light to excite the compound of interest and monitor the intensity of light emitted at a specified emission wavelength, with the emitted light proportional to the concentration of the analyte. Most fluorometers utilize monochromators or filters to select wavelengths. Turner Designs TD-360 Designed for integration with HPLC systems, Gilson provides the Model 121 and 122 Fluorometers, which are purportedly simple to use. Both systems utilize interchangeable filters and flow cells. The new Model 122 allows programming of 10 method files with up to 64 steps per method. A built-in thermistor evaluates bench temperature and compensates for temperature variation via feedback from the fluorometer's central processing unit. The 121 comes standard with a 9 ml flow cell, while the 122 is equipped with a 16 ml flow cell. The DyNA QuantTM 200 from Pharmacia Biotech is designed specifically for the accurate quantitation of small-volume samples with low DNA concentrations using the Hoechst 33258 dye. This dye binds preferentially to double-stranded DNA, thus allowing accurate quantitation even in the presence of RNA, proteins, and/or nucleotides in cell lysates. The DyNA Quant 200 can accept one standard cuvette or capillary cuvette. St. John Associates provides the FLUORO-TEC Fluorometer as a test tube model or a flow cell system. Cuvette adapters are available for capillary tubes, round tubes, and a rectangular spectrofluorometer cell. The FLUORO-TEC is only 41/2 inches wide and weighs only 8 pounds. This machine is suited for a wide range of fluorescence-based methods. The 10-AU-005-CE Field Fluorometer from Turner Designs is a rugged, field-portable instrument that is capable of continuous-flow or discrete sample analyses. The unit is equipped with removable filter paddle, and the O-rings of the filter paddles are compression-sealed, ensuring a watertight design. With internal data logging, up to 64,800 data points can be stored in the instrument's memory and saved, even when the instrument is not continuously powered. Data can be downloaded, stored in ASCII format, and imported into a spreadsheet format for manipulation. Also available from Turner Designs is the TD-700 Laboratory Fluorometer, designed as a compact, low-cost benchtop fluorometer. Automatic range optimization allows the automatic setting of range, gain, and resolution, eliminating operator error due to working in the wrong range. A multiassay filter cylinder houses eight interchangeable filters for four separate applications. The TD-700 accommodates test tubes, square cuvettes, and optional 100 ml minicell or 9 ml microcell adapters. The Kinetics software outputs data to a computer or printer at selectable intervals. Brand new from Turner Designs is the TD-360 Mini-Fluorometer, a dedicated, single-cuvette flurometer that features a unique quick-change filter holder, easily-accessible lamp module, RS232 serial interface, and ability to store up to five calibration settings. A variety of compounds can be measured in standard square cuvettes or 100 ml disposable minicells using application-specific optical filters and lamp sources. Sensitivities as low as 500 picograms/ml DNA using PicoGreen are easily achievable. Bio-Tek offers the FL600 Fluorescence and Absorbance Microplate Reader. The FL600 can scan 6- to 384-well microplates (including Terasaki plates) and its dual optical channels provide for software selection of top or bottom epifluorescence measurement. Four probe sizes, high resolution probe movement and a large optical chamber enable reading of practically any container, including PCR tubes. Six position and independently controlled filter wheels provide many filter combinations for a variety of probes and dyes. Temperature control up to 50°C supports kinetic capabilities. The new KC4TM for WindowsTM data reduction program features extensive graphics and data reduction capabilities and is simple enough for beginning users to operate. The FLUOstar from BMG LabTechnologies can measure a variety of plate formats. By defining the locations of the corner wells, along with the number of rows and columns, the FLUOstar can measure any plate format, including 384-well plates and Terasaki plates. The FLUOstar's software- selectable flash control allows both standard and time- resolved fluorescence applications. PC software provides instrument flexibility, and available templates include replicates, standard curve regression, graphics, and unknown sample calculations. Bio-Tek Instruments FL600 The flexible FluoroliteTM 1000 Fluorescence Detection System from DYNEX Technol-ogies can read 96-well plates as a stand- alone instrument, or 6- to 96-well plates when under PC control. The Fluorolite 1000 is also capable of reading 35 mm petri dishes and membranes for dot blot assays. A user- selectable excitation and emission filter set provides more than 75 different filter combinations. The Windowsª-based BioLinx Assay Management Software provides control over all unit functions (except voltage). BioLinx provides control over assay functions such as template design, difference matrix, ratio matrix, curve fits, threshold formulas, histograms, sample identification, plate identification, and report formatting. BMG Lab Technologies FLUOstar The Spex Fluorolog-3 from Instruments S.A. is one of the more unusual machines encountered in this profile. This self- calibrating spectrofluorometer is modular, enabling users to choose a source, spectrometer, sample compartment, detector, and accessories that match the wavelength range, frequency domain, or physical characteristics and parameters of samples. When equipped with the MicroMax microplate reader, fluorescence data are acquired at a speed of about 100 samples per minute. For advanced operations in Windowsª, DataMax-STD software allows users to set up different prompt sophistication to suit varying levels of experience-novice, intermediate, or expert. Automated sampling accessories include polarizers, sample changers, plate readers, grating turrets, and more. Instruments S.A. also offers the Fluorolog3-Tau3 Lifetime accessory, which allows picosecond to microsecond fluorescence lifetime measurement capability in the frequency domain. This accessory is ideal for research in energy transfer and dynamic depolarization. Another powerful research tool from Instruments S.A. is the Fluoromax-2. The Fluoromax-2 is a self-contained, benchtop spectrofluorometer with high speed and sensitivity, ideal for a wide range of analytical fluorescence measurements. Instruments S.A. Fluorolog-3 The BioLumin 960 from Molecular Dynamics is a fluorescence and absorbence microassay reader. According to Molecular Dynamics, the BioLumin 960 can read fluorescence and absorbence on one microplate in 12 seconds. Temperature control, from ambient to 45°C, is a standard feature of the BioLumin 960. XperimentTM software for MacintoshTM lets users design experiments by arranging icons on the computer screen in the desired sequence. Xperiment also facilitates viewing of data summaries and kinetic assay plots, plotting of standard curves, calculation of unknowns, and printing of organized reports. Packard Fluorocount Packard's FluoroCountTM Microplate Fluorometer can accept 6-to 96-well microplates and can excite and measure samples from above or beneath microplate wells. Excitation and emission filter wheels (with up to four filters each) let users sequence four wavelength combinations for each plate reading. The HTS 7000TM Bio Assay Reader from Perkin-Elmer was designed specifically for rapid turnaround of nonradioactive, photometric analyses on large arrays of small volume samples. The WindowsTM-based HTSoftTM instrument control and data analysis software provides simple software options which allow selection of stored user methods for top and bottom reading fluorescence or through-well absorbance measurements down to 260 nm. No hardware reconfiguration is necessary to change measurement modes and all plate types and styles from 6 to 384 wells can be added to the user library. From DNA and protein quantitation, cell function assays, kinetics, and ELISA assays, the versatility of providing three instruments in one platform allows the user to perform virtually any fluorescence- or absorbance-based biochemical assay on the HTS 7000. Molecular Dynamics BioLumin 960 Select between fluorescence or low UV absorbance microplate reading with the SPECTRAFluor from TECAN. With fluorescence, measurements of 6- to 384-well microplates and Terasaki plates are possible. Dual optical channels allow measurements from above and below the plate. Thermal incubation, orbital or linear shaking, gain control, and user-defined flashes per well can easily be controlled within the PC or MacintoshTM software program. Tecan US SPECTRAFluor The 1420 VictorTM from EG&G Wallac is a multilabel counting instrument, combining a time-resolved fluorometer, photometer, and luminometer in one instrument. The Victor accepts 6- to 384-well microplates, Terasaki plates, a petri dish, PCR tubes, and although it is a small benchtop unit, it can be integrated into a robotic system. A unique stacking system allows walk-away loading of up to 20 plates at a time. Also, the plates can be barcoded for easy tracking. The VictorTM runs on WindowsTM 95-or Windows NTTM compatible computers. Chart The author can be reached online at sbeck@the-scientist.com.

Luminometers and Fluorometers

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