Illuminating Images: The World of Phosphor Screen and Fluorescence Imaging Systems

Date: February 16, 1998 Chart 1 Chart 2 nyone tired of mixing darkroom chemicals should read this article. The modern generation of instruments for viewing both radioactively and fluorescently labeled samples can save you countless hours of time and provide quantitation only dreamed of by researchers in the past. The products to consider are phosphor screen and fluorescent imagers. These handy devices offer numerous advantages over film autoradiography: Sensitivity: estimates of sensitivity are anywhere from 10 to 100 times greater than that of X-ray film. Range: the dynamic range of some imagers is as great as five orders of magnitude, compared with two at best in the case of film. Time: time savings come in a number of areas-preparing chemicals, exposing film, processing film, and reexposing film if the exposure is not quite right. Ability to quantitate data: this is what really puts these machines in the hearts and budgets of researchers-no more cutting out bands, weighing, measuring, scanning films, and struggling with issues of linearity of film, background correction, and shelf life of chemicals. For the most part, these advantages apply to imaging and analyzing fluorescence as well, for which there was little beyond color photography (except very sophisticated and expensive image processing instruments) for documenting and analyzing this kind of data. There are currently at least three broad categories of instruments: those that image only radioactivity (Fuji Medical's BAS Series, Molecular Dynamics' PhosphorImager SI, and Packard Instrument's CycloneTM and InstantImager"), those that measure only fluorescence (Molecular Dynamics' FluorImager 595), and those that do both, or do more than one kind of detection, called a variety of names. Further confusing the picture is the fact that non-radioactive labels now come in different types: the old standby fluorescence, chemiluminescence, which until recently was the preferred tag for membrane detection, and more recently chemifluorescence, which was designed to combine the advantages of fluorescence and chemiluminescence. Some phosphor screen imagers (Bio-Rad's GS models) can also be used for chemiluminescence, and others (Fuji FLA 2000 and Molecular Dynamics' Storm") for chemifluorescence. Among the fluorescence imagers, multiple-use machines include Hitachi's FMBIO" II and Raytest's DIANA, which were designed for detection of chemiluminescence, and fluorescence, and the newest entry in this field, Bio-Rad's Fluor-S MultiImager, which detects chemiluminescence, chemifluorescence and single or multiple fluorescent signals. Reprinted with permission from Molecular Dynamics, Inc. The current generation of imagers has combined the technologies of autoradiography and spectrophotometry to create instruments that both detect and quantitate signals emanating from biological materials. Exactly how they do this varies somewhat, but many use laser scanning. In this process, a laser is moved over a sample, exciting a phosphor embedded in the storage screen or fluorescence emanating from the sample itself. (Only one machine, Packard's InstantImager, images radioactivity directly; all the rest use phosphor storage screens, similar to intensifier screens used in conventional autoradiography, that accumulate charge from the radioactive sample over a period of time before being imaged.) The signal is collected on a photomultiplier tube (PMT), the data from which is converted from analog to digital for the eventual construction of an image using software packages provided by the manufacturer of the instrument. Different companies have employed different lasers, some proprietary, but the most commonly used is helium/neon, or He/Ne, which excites the phosphor used in most screens. Some companies specializing in fluorescence imaging use PMTs while others use cooled CCD cameras to collect the signal from laser-excited fluors. Instead of scanning, they collect the entire image at once-a process that is faster and potentially more sensitive than scanning (since the machine can add images together) than scanning, but provides lower resolution. However, for most applications (gels, blots, and plates) even the poorest resolving imager is adequate, and in many situations, time is a more important factor. In addition to the simple laser scanning process described, some other configurations have been developed-multiple lasers, rotating mirrors, mounts that fix the laser and detectors in a constant position-each of which provides different and particular advantages, as detailed in the following machine descriptions. Some of the features that distinguish one imager from another are sensitivity, speed, and resolution, and depending on your lab's applications, each of these issues will carry different weight. Nothing beats the straight phosphor screen imagers for sensitivity, some going down to < 1 dpm/mm2. Therefore, for experiments in which very low activity is expected, these would be the machines to consider. On the other hand, for some lab applications, particularly those involved with tissue localization or gels with closely spaced bands, resolution, rather than signal intensity, is the limiting factor. The earliest imagers were not highly resolving (Packard InstantImager excepted), but among the present group of instruments, a number are claimed to resolve at 50 æm (with one instrument, the Fuji BAS 5000, coming in at 25 æm), which is well below what is needed for most applications (80-100 æm). The single most important factor in choosing an instrument will be the kinds of signals you anticipate detecting. If you foresee using only radioisotopes, then the choices are obvious. A feature that distinguishes between the straight phosphor screen imagers is the choice of screens, which can differ both in chemistry and thickness of phosphor layer. Both Fuji and Packard offer a variety of screens for different applications, while Molecular Dynamics relies on Kodak for their phorphor screens, though the chemistry is roughly the same. Bio-Rad, on the other hand, uses an entirely different chemistry from the others. Similarly, if you anticipate using fluorescence only, the choices are limited to Hitachi's FMBIO and Molecular Dynamics' FluorImager 595. The principle difference between these two instruments is the laser, with Hitachi choosing one with a fairly high wavelength of excitation, and Molecular Dynamics a dual line argon laser. Once you move into multiple-use instruments, the field becomes cloudy and you will have to spend some time mulling over options of detectors and detection devices. For example, several of the multiuse instruments, such as Bio-Rad's MultiImager and Raytest's DIANA II, use CCD cameras for signal collection. Bio- Rad's GS models and Molecular Dynamics' Storm offer laser scanning phosphor imagers that will work with chemiluminescence and chemifluorescence, respectively, so the decision would rest on the nature of the probes at your disposal. Also note that the phosphor screens employed by these two manufacturers have different chemistries with different properties in terms of resolution, background, and light sensitivity. The Bio-Rad MultiImager offers the largest range of use-encompassing chemiluminescence, fluorescence, and chemifluorescence and the capacity to detect multiple fluors as well, but is limited in dynamic range and resolution. Fuji and Raytest also offer a range of uses; autoradiography as well as fluorescence. With machines roughly comparable in their abilities, often the deciding factor will revolve around people issues: ease of use, technical support, and training. In the case of phosphor screen imagers, ease of use rests largely on the screens. Some require special handling and darkrooms or dark boxes, while others have been designed for use in normal room light. Whatever instrument you use, the file size generated may also be a parameter that will affect the ease of use in your laboratory, as very large files can require constant vigilance in maintaining hard drive space on computers, particularly when there are multiple users. Aside from checking with the manufacturer, internet chat rooms offer an avenue for obtaining information on products potentially from users around the world. Imager Group Fuji Medical Group USA BAS Series Fuji offers a series of Bioimaging Analysis Systems (BAS) dedicated to autoradiography applications. The newest in the series, the BAS-1800, has been upgraded for cost performance while requiring less space than its predecessor, the 1500. The BAS-1800 maintains Fuji's standard for quantitative accuracy, resolution, and linearity of data. Using sensitive Fuji Imaging Plates, which have a phosphor layer on a flexible support, the BAS-1800 provides high- resolution scans (50 æm resolution) in three minutes. Fujis Imaging Plates come in different sizes and are sensitive to different isotopes, including neutrons. Applications include gels, blots, in situ hybridization, receptor binding assays, TLC, and whole body autoradiography, as well as applications in physical and meterial sciences. Molecular Dynamics PhosphorImager® SI For filmless autoradiography, Molecular Dynamics offers the PhosphorImager SI, which is sensitive to the commonly used radioisotopes 14C, 3H, 125I, 32P, 33P, 35S, and others. Using a helium/neon laser for excitation, phosphorescence is collected by a fiber optic bundle and channeled to a photomultiplier tube. The resulting data, which retains positional information, is digitized with 16-bit precision to form an image. This machine measures over four orders of magnitude. Molecular Dynamics provides durable screens that require no special equipment for handling or erasure. The PhosphorImager SI comes with ImageQuant software and documentation for Mac or PC, with which 1-D analysis of density profiles (peak area integration) and volume quantitation of signal intensities can be determined. Also available are software programs for fragment analysis, genetic typing, DNA scan, and phoretix (automatic 2-spot analysis). Typical applications include DNA sequencing gels, DNA blots, CAT assays, protein blots, X-rays, and whole body autoradiography. Molecular Dynamics provides technical support and a one-year warranty. The company has a guide to fluorescent imaging applications available on their web site, and in addition supports a florescence users group accessible through the internet. Instantimager Packard Instrument Company CycloneTM InstantImager® Designed for high-resolution scanning of radioactive samples, the Cyclone is a storage phosphor scanner that offers fast and high-resolution scans using confocal optics and a helical scanning system. The laser and optical system are mounted on the same platform, which moves down the screen as the screen rotates on a carousel, keeping the laser excitation and light collection optics in a consistent position relative to the surface of the screen. This unique design eliminates some of the problems associated with galvanometer-driven mirrors directing the beam. Cyclone can do simultaneous exposures for low-activity samples and outputs publication-quality images. Packard offers a variety of screens optimized for different applications: multipurpose screens (MP), which are moisture proof; superresolution screens (SR), which have a thicker layer of phosphor crystals for greater efficiency of capture; supersensitive screens (ST), which have fine crystal grains for high resolution; and tritium-sensitive screens (TR), which are uncoated so the low energy of tritium can be detected. The Cyclone comes with OptiQuant, a Windows 95-based software package, which has scan control software for specifying resolution and scan area during image acquisition, on-screen image display functions, and advanced palette control features including linear, log, sigmoid, or customized scaling for publication- quality images. InstantImager® is a one-step system that does direct counting and imaging of samples without cassettes or screens. InstantImager counts the entire sample area simultaneously without scanning or stepping and provides real-time results as soon as the activity begins. The patented detector is a multiwire chamber with microchannel array converter (MICAD) that localizes the radioisotopic events and provides spatial resolution adequate for most life science applications. All samples within the 20 x 24 cm sample area can be quantitated simultaneously by microchannels that localize and amplify the electrons from the ionization events. InstantImager uses InstantQuant software. FMBio® II Fluorescence Imaging System The Fluorescence Imager Group Hitachi FMBIO® II Fluorescence Imaging System Hitachi has developed the FMBIO II to improve the accuracy and efficiency of laser scanners. Using a solid-state green YAG laser, the laser's optical unit is moved across the sample as the laser is directed onto the sample via a polygon mirror rotating at high speed. The resulting fluorescent light signals are collected by an optical fiber array, filtered through band pass filters, and converted to electrical signals by two photomultiplier tubes. Analog-to-digital conversion completes the process, generating an image for interpretation and analysis. FMBIO provides a solid state laser that allows excitation and detection of a variety of fluorescent dyes and chemilfluorescent substrates. Dual PMTs allow simultaneous detection of two fluorophores, further enhancing the system's utility. Applications include differential display, genotyping, array analysis, DNA sequencing, agarose gels, membrane-based applications, single strand conformation polymorphism (SSCP), and microsatellite analysis. touts high throughput, high accuracy, high resolution, and application versatility as among the advantages of the FMBIO II Fluorescence Imaging System. Molecular Dynamics FluorImager 595 The FluorImager 595 is a multipurpose dual-excitation laser-induced fluorescence scanner for electrophoresis gels, membranes, thin layer chromatography (TLC) plates, and microplates. Fluorescent sample components are read directly in the gel or plate with no drying or exposure steps required. The FluorImager 595 supports a wide variety of multiple fluorescence labeling protocols. These include enzyme substrate labeling, covalent prolabeling of nucleic acids and proteins, and pre- or post-separation labeling of DNA with intercalating dyes. The FluorImager 595 includes software that controls scanning, multiple color separation, image display, and quantitative tools for data analysis and produces final reports. Bio-Rad Molecular Imager FX System Bio-Rad Model GS-525 and GS-505 Molecular Imager Systems Fluor-S MultiImager System Molecular Imager FX System Bio-Rad's Model GS-525 and GS-505, which differ only in scanning area, are designed for both chemiluminescence and autoradiography imaging and quantitation. They provide sensitivity 10 to 100 times greater than that of X- ray film, a dynamic range of five orders of magnitude, the ability to detect strong and weak signals, and short exposure times. The Bio-Rad units are sensitive to all gamma emitters, high-energy beta emitters, weak beta emitters, and tritium. Chemiluminescent substrates detectable are horseradish peroxidase- activated substrates and alkaline phosphatase-activated substrates. The exposure platform is a light-tight cassette that accommodates blots, gels, slides, TLC plates, and microplates. Light-sensitive screens respond to 400-500 nm light with a dynamic range of four orders of magnitude. The Model GS-525 is designed for DNA sequencing-sized samples (35 x 43 cm), while the GS-505 is for smaller platforms (20 x 25 cm). Bio-Rad uses a patented fiberoptic/laser scan ning design to capture signals and imaging software that allows transfer across PC and Mac platforms. Data and images are exportable as well to Microsoft® Excel and Adobe Photoshop programs. The Bio-Rad Fluor MultiImager System can be used for a variety of applications: single-color fluorescence detection, multiple-color fluorescence detection, true chemiluminescence detection, chemifluorescence, colorimetric detection, and gel documentation. Bio-Rad's unique UV and white light epi-illumination and scanning illumination technology provide a greater integrated excitation than laser-based excitation, resulting in sensitivity down to femtomole amounts of end-labeled DNA or picogram amounts of stained DNA. The MultiImager has six variable filter positions and provides multichannel acquisition and display for rapid image analysis. The new Molecular Imager FX system provides the ability to perform the widest range of imaging applications including: radioactive imaging using a choice of phosphor screens, single and multi-color fluorescence via direct laser excitation, and chemiluminescence using phosphor screen technology. The key to this instrument's flexibility is the dual PMTs and dual-wavelength internal laser system. For applications requiring additional laser lines, an optional external laser can be added for unparalleled fluorescence flexibility. This field upgrade guarantees that the Molecular Imager FX system will allow your laboratory to take advantage of any change in research focus or future development in fluorescence technology. LAS-1000 Luminescent Image Analyzer Fuji Medical Systems USA LAS-1000 Luminescent Image Analyzer FLA-2000 The LAS-1000 single system features Fujifilm's new cooled 1.3-million-pixel CCD camera to capture chemiluminescent, fluorescent, and chemifluorescent images at high resolution and high sensitivity. This camera can handle continuous exposure up to one full hour and has one-square-inch chips with each pixel covered with a micro lens. Exposure time can be optimized by checking the monitor image using one-by-one exposure mode, and the image is promptly displayed. Capture of even faint-light images is possible in ordinary room light using the LAS-1000 "Intelligent Dark Box." The LAS-1000 has a wide dynamic range, as the large pixels of the CCD chip enhance the relationship between the incident rays and the generated electric charge; the Fujinon URF20L lens allows for more efficient light collection. Applications include the chemiluminescent substrates CDP -Star® and ECLTM; fluorescent substrates ethidium bromide, SYBR® Green I and II, and FITC; and the chemifluorescent substrate Atto PhosTM. The LAS-1000 accepts membrane or gel samples. A dual-purpose fluorescence and radioluminography scanner, the FLA-2000 has two separate lasers: a newly developed Second Harmonic Generation laser and a He/Ne laser. This technology enables a broad range of applications in the life sciences, including the common fluorescent assays-SYBR Green I staining of DNA, analysis of high-density arrays, SYPRO® Orange staining of protein gels-as well as autoradiography and densitometry. The FLA-2000 incorporates Fuji's proprietary Phosphor Imaging Plate technology, which provides sensitive and efficient image processing. Storm Molecular Dynamics Storm® The Molecular Dynamics Storm system for gel and blot (Southerns, Westerns, and Northerns) analysis unites proven PhosphorImager® system technology with two nonradioactive fluorescent imaging modes of red and blue fluorescence. Now researchers can make the move to emerging nonradioactive gel and blot analysis methods without giving up proven radioisotope techniques. Storm provides users with a wide range of chemistries to choose from for all their fluorescent applications. Storm is capable of scanning storage phosphor screens plus fluorescent gels, chemifluorescent blots, some chemiluminescent blots, and direct blots (for example Cy5, Cy3, or fluorescein labeled) in the 35 x 43 cm scanning area. Storm includes ImageQuant for Windows NT or Macintosh to assist in analysis of electrophoretic separations, dot blots, slot blots, microplate assays, TLC assays, and plaque assays. ImageQuant provides a point-and-click environment for fast analysis of qualitative and quantitative assays. With tools for accurate volume integration, ImageQuant generates accurate lane profiles even for curved or slanted lanes. Peaks in the lane profile are automatically located and integrated. Volume and area integration results are automatically transferred to MicrosoftTM Excel for further analysis and reporting. DIANA II Raytest DIANA II: Digital Image Analysis Acquisition The DIANA from Raytest is a unique imaging detection system designed for a wide variety of nonradioactive applications. It has a cooled CCD camera with a light- tight darkroom and height-adjustable sample table. Three different probe holders are available for the sample table: a metal plate for chemiluminescence detection, a UV transparent Plexiglas plate for fluorescence detection, and a UV-to-visible light converter plate for colorimetry detection. DIANA's cooled CCD camera is intended and designed for low light levels required for sensitive chemiluminescence detection. DIANA is suitable for all standard applications in gel electrophoresis such as ethidium bromide, SYBR Green I, and FITC. In addition, all color staining used in bioimaging can be detected and documented by DIANA's cooled CCD. The DIANA features a three-log dynamic range and excellent signal-to-noise ratio, 12-bit digitalization directly in head for accurate quantitation, and a software-controlled filter wheel for convenient filter changes. AIDA software is supplied for evaluation and documentation of images obtained with digital CCD cameras and flat-bed scanners. Typical applications are one- and two-dimensional image analysis, and migration distance measurements for determining molecular masses, DNA sizes, isoelectric points, and RF values. The author acknowledges the assistance of Jim Bolen, Manager of the Flow Cytometry and Laser Scanning Core at the City of Hope National Medical Center. Chart 1 Chart 2 Laura DeFrancesco can be reached at ldefrancesco@the-scientist.com.

Illuminating Images: The World of Phosphor Screen and Fluorescence Imaging Systems

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