If you really think about it, data is only as good as the ability to acquire, quantitate, and compare various points that may be significant to the underlying observation. During the "historic" era of science most analyses were performed by visually evaluating a series of films or other forms of data and making side-by-side comparisons and interpretations. Conclusions were, for the most part, qualitative in nature, and rarely could true quantitative values be ascribed to particular bands or spots. As a result, one usually relied on a vocabulary of creative adjectives to describe changes of the data points.
With the application of electronic and imaging technologies, significant advances have been made in the ability to collect and quantitatively analyze data. Laboratories now have the capability of acquiring and quantitating data points, making representative comparisons using hard numbers, and consequently making more accurate predictions based on more than just what a graduate student's adjectives allow. Current hardware components also allow data to be collected from an unprecedented range of sources: signals from fluorescent detection labels, data from colorimetric assays, and the old reliable black-and-white images of autoradiograms.
Coupled with the ability to manipulate images captured by such devices, computers and software have expanded the range of analysis capabilities. Electronically compensating for a smiling gel has allowed for more accurate estimation of molecular weights; subtracting background similarly provides for more accurate estimation of quantities of an amplified band. Even enabling the use of overlay lanes has revolutionized the ability to compare and interpret data. Perhaps as important, while the cost of these systems may be more expensive than what we are used to paying for a land camera, the availability of electronic files allows for inexpensive printouts, the ability to call up a file and reanalyze the data, or the preparation of a figure that can be inserted into a manuscript or presentation. Together these features create systems which are very cost effective.
So digital imaging and image analysis seems to have piqued your interest? As the accompanying profile demonstrates, the number of companies and products in gel documentation has expanded significantly. Decisions on which system is best are clearly a matter of your budget, the nature of your data (chemiluminescent, stained gels, etc.), and whether you are interested in basic documentation or lean more heavily toward quantitative analysis. A comprehensive dissertation on detail aspects of hardware and software is beyond the scope of this article. With this in mind, the following is a brief description of various aspects of hardware components that you might want to keep in mind when looking more thoroughly into company literature. We hope to visit the question of software in a future issue.
There are two basic hardware formats for acquiring or imaging data electronically that you will encounter in commercial documentation and analysis packages. Both of these formats has certain advantages, and there may be some benefit to having access to both to accommodate a laboratory's overall needs. At one end of the spectrum are camera-based systems. These systems provide a method of collecting images from a wide variety of formats ranging, for example, from ethidium bromide-stained gels, to standard autoradiographs, to the real-time capture of chemiluminescent emissions. At the other end are scanners that typically are used for dry, flat data formats. These range from high-end transmission/reflectance flat-bed scanners to simple handheld models.
Probably the most common camera systems are CCD (charge-coupled device) cameras. These cameras, usually used in conjunction with a light source (white or UV light), are good all-round systems suitable for general imaging work. For routine documentation that basically requires only capturing an image, almost any system will work. More sensitive camera systems are, however, required for systems that do not utilize a light source, for example, when the requirement is to capture emissions directly from chemiluminescent assays. For these applications, the sensitivity of the hardware is an important issue. A camera's sensitivity, or ability to function at low light levels is measured by a camera's lux rating. Cameras with a low lux rating are better able to collect very low light emissions and are therefore recommended for applications such as chemiluminescence.
CCD cameras operate by collecting photons on a chip. Over a given period of time a discrete charge is collected on the chip which is translated into a digital signal correlated to signal strength. Some cameras offer the option of integration, which is the ability to accumulate the charge over a greater period of time. This feature can be thought of as a method of varying exposure levels. Ideally, the longer the integration time, the better-particularly when capturing emissions from chemiluminescent data, for example. However, with increased time, there is an increase in the level of noise, which will degrade the quantitative data you are attempting to acquire. Integration can be performed by the camera itself or may be incorporated in the companion software. It is generally agreed that on-chip integration is preferable for higher level performance. Also remember that a camera with a low lux rating will require less integration since it is more efficient at gathering a signal. The ideal target then-and this can be where your budget goes out the window-is a camera with a low lux rating and the ability to perform integration.
Some cameras are described as "cooled" CCD cameras. For all CCD cameras, noise becomes the enemy of your ability to discriminate. Cooling reduces background noise. Whether this feature is necessary is subject to debate.
One other feature to be aware of is camera resolution. In general, the larger number of pixels in a CCD camera, the greater the resolution. Note, however, that as more information is collected, the file size of the image will increase. High-resolution imaging will therefore rapidly increase the demand for more powerful hardware.
For various applications, cameras need to be equipped with special filters to accommodate different working wavelengths. Typically these filters are mounted on the camera in front of the lens. Filters for UV work, for example, diffuse the UV rays so that the emission source (light box) is not "visualized" by the camera. It is important to consider the various applications (SYBR-green, ethidium bromide, etc.) in your lab and have the appropriate filters available.
Finally, one should be aware of other camera systems on the market. CMOS cameras are lower cost systems that have detection elements less sensitive than their CCD cousins. Consequently, these cameras have historically had higher signal-to-noise ratios, although newer generations of these cameras have narrowed this gap. Additionally, CMOS systems do not integrate as well as CCD cameras. Digital photographic cameras are also beginning to make an appearance on some laboratory benches. Kodak Scientific Imaging offers the only digital camera-based system in this article. An inexpensive alternative for routine documentation, this camera does not have the ability to perform integration. However, it can be used for any laboratory application where full color images are required-it is not dedicated to gel documentation.
Cameras aside, there are other methods of capturing data for evaluation. The field of scanner-based imaging systems is less crowded than that of cameras. Entry level scanner systems can be significantly less expensive than CCD-camera based systems. Many scanners are desk-top, flatbed machines that can accommodate large format autoradiographs. At the other end of the spectrum are useful little handheld scanners with hardware-based contrast adjustments. This feature allows the optimization of the image prior to data acquisition, and does not rely on subsequent software adjustments.
From a performance perspective, in comparision to cameras, scanners have greater uniformity of illumination. Much like photographic-emulsion cameras, CCD lenses are weighted; that is, they have their best response in the center of the image. A good scanner, however, will provide a broad, linear dynamic range over a range between 0.01 and 3.0 OD. Scanners also have an advantage in resolution, with many capable of achieving 1200dpi with 12-bit data. This translates into better spatial and grayscale resolution of data. Scanners perform particularly well with autoradiographs, blots and Coomassie or protein stained gels. Scanners can be used to image nearly any format, even including wet gels for some models.
One limitation of scanners, however, is the inability of most to accomplish UV imaging of ethidium-bromide gels. A small number of scanners will operate in both the transmitted and reflected light modes, however, making it possible to image transparent and opaque samples. As a result, many laboratories may choose to have both CCD-based and scanner equipment available.This strategy allows the user flexibility in acquisition of all types of images.
The above is meant to provide only a brief introduction to the features to look for in an image analysis system. It is not a comprehensive technical analysis, and serves only as a primer to allow you to begin to interpret literature from various companies. Of course, hardware is only one component of an analysis system. Your choice of software is the second step in your ability to analyze data. Keep in mind that the quality of the input will effect the quality of your analysis. It is therefore wise first to evaluate your hardware requirements.
A discussion of the features and capabilities of companion software is beyond the scope of this introduction. These products will be the subject of a future article.
Advanced American Biotechnology (AAB)
Gel Documentation System and Gel Doc I
The Gel Documentation System from AAB is comprised of a Pentium computer, time integration video camera, zoom lens, UV filter, dark room, dual transilluminator, frame grabber, and universal software for gel quantification and analysis. The Gel Doc I features a 0.03 video camera, frame grabber, lens, UV filter, dark room, and video printer. AAB also markets a wide range of software products for virtually any gel application. (714-870-0290 or www.members.aol.com/aabsoft)
AlphaImagerTM, ChemiImagerTM, AlphaScanTM, and AlphaDocTM Digital Imaging Systems
AlphaImagerTM imaging systems from Alpha Innotech Corporation feature non-vineatting zoom lens, fluorescent filters, customized computers, and AlphaEaseTM imaging software. The ChemiImagerTM utilizes a proprietary 3-stage, thermoelectrically cooled CCD down to -40¡C. The cooled CCD allows for real time display and is ideal for white light, fluorescent, and low light applications such as gels, films, membranes, plates, and chemiluminescent samples. The AlphaScanTM scanning densitometer system provides a high resolution and dynamic range scanner for transmission and reflectance samples. All systems incorporate the AlphaEaseTM software which has over 150 image processing and analysis features and is available for Windows 95, NT, and PowerPC. The AlphaDoc is an entry-level system designed to reduce the cost of film for routine gel documentation. (800-795-5556 or www.alphainnotech.com)
Ana-Gen Technologies ImageDOC System
Ana-Gen's ImageDoc System is a complete entry-level system. The basic system consists of a high-resolution CCD camera with manual focus, a high-resolution monochrome monitor, a 256 gray scale video printer, a low profile darkroom cabinet, a UV transilluminator, a camera filter for ethidium bromide gels, and all necessary stands and cables. The system is completely modular and can be upgraded. The 9" monitor enables users to preview real-time images prior to printing. System options include a darkroom cabinet with a manually adjustable slide camera mount, a dual white light/UV transilluminator, a zoom lens, an integrating camera, and a frame grabber circuit board with image storage and text annotation software. (800-654-4671 or www.ana-gen.com)
GelPrint 1000i, GelPrint 2000i, GelPrint Plus, and GelPrint Ultra
BioImage offers sytems capable of a wide range of documentation and analysis. The GelPrint 1000i features a CCD with fixed focal lens, 256 gray scale analog video printer, and is a dedicated documentation system. The GelPrint 2000i has a CCD with zoom lens, 256 gray scale digital graphic printer, and allows for digital archiving through a floppy drive and has network capabilities. Both the GelPrint Plus and GelPrint Ultra run off a PC platform, and come with a 600 dpi laser printer, dark room enclosure, and comprehensive software packages. The GelPrint Plus has an 8-bit cooled camera with zoom lens, while the GelPrint Ultra has a 10-bit cooled camera. (800-BIO-IMAGE or email: firstname.lastname@example.org)
BioDoc I and BioDoc II Video DocumentationSystems
These two systems from Biometra feature CCD cameras and "on-chip" integration. The simple BioDoc I allows direct documentation with a video thermoprinter. The newer BioDoc II features a practical softkey keyboard, 100 MHz Pentium PC, color monitor, choice between analog or digital thermoprinter, and optional integration into a local network. Images can be stored and transferred on the BioDoc II, and a variety of software packages are available for gel analysis. (551-50-68-6-0 or www.biometra.de)
Gel Doc 1000, Insta Doc I and Insta Doc II
Bio-Rad offers a complete line of gel documentation systems. The Insta Doc I is the lowest cost starter system. The Insta Doc II includes a complete darkroom cabinet with transilluminator. The Gel Doc 1000 is a complete analysis system for documentation, archiving, volume and molecular weight analysis. All systems utilize an 8-bit high-resolution CCD camera and are upgradeable from one to the other at any time. Bio-Rad also offers a complete line of image analysis software for RFLP, 2D, and DNA sequencing. (800-4-BIORAD or www.bio-rad.com)
Eastman Kodak Company
Kodak Digital Science EDAS 120 System and BandScanner 1D System
The EDAS 120 from Eastman Kodak features the DC120 Zoom Digital Camera. This camera has auto/manual flash, aperture, and shutter speed, an LCD preview/review screen, and built-in as well as removeable memory. The BandScanner 1D System digitizes gels and blots using the 600 dpi Hewlett Packard Scanjet 6100. Both systems include Kodak Digital Science 1D Image Analysis Software which has built-in acquire modules for image capture, and facilitates image adjustment, quantitative analysis and non-destructive annotations using Power Macintosh or Windows 95/NT platforms. Printed output is accomplished using the Kodak Digital Science SP700 printer, which uses dye sublimination technology and prints 24-bit continuous tone color as 4" x 6". (800-225-5352 or www.kodak.com)
FOTO/Analyst MiniVisionary, Visionary Eclipse, Archiver Eclipse, and Investigator Eclipse Systems
FOTODYNE offers a wide range of products for gel documentation and analysis. The MiniVisionary Apprentice and Visionary systems offer simple electronic documentation. The Archiver system allows storage of up to four images on a floppy disk and production of instant thermal prints. The Investigator is FOTODYNE's most powerful electronic imaging system, offering the capabilities of thermal prints, diskette storage, and computer assisted image analysis. All FOTODYNE systems are CCD-based. (800-DNA-FOTO)
Speedlight Jr. GDS, Speedlight GDS, and Speedlight Platinum GDS
The Speedlight systems from Lightools Research are stand alone and do not require a PC for operation. These systems are CCD-based and come with a 256 gray scale thermal printer. The Speedlight Platinum was designed for centralized data collection and decentralized off-line data analysis. The floppy disk provides a conduit to transfer digitized data from the GDS to a computerized workstation. (760-632-0677 or www.lightools.com)
NucleoVisionTM 420, 530, 760, 830, and 920 Systems
NucleoTech provides numerous systems for gel documentation and image analysis, including compact "documentation systems"; systems for image processing, archiving, and analysis; and custom systems. CCD camera options include non-cooled, (two-stage) Peltier-cooled, and a color CCD that can be used for microscopy applications. Using a PC or Macintosh platform, the GelExpert software suite features true 32-bit architecture programming, native for Windows 95/NT 4.0 or Macintosh. (510-785-9701 or www.nucleotech.com)
Pharmacia Biotech ImageMaster VDS
The ImageMaster VDS is a self-contained, stand alone sytem featuring a digital video CCD with signal integration occuring at the chip level. Also included with the ImageMaster is a zoom lens, filter, monitor, transilluminator, UV filter, and built-in thermal printer with proprietary polymer film using emulsion chemistry. The ImageMaster does not require a PC for operation, and can detect 20 picograms/band for ethidium bromide-stained DNA in an agarose or polyacrylamide gel in less than two seconds. Optional with the ImageMaster VDS is a computer interface board, image capture software, and image analysis software. (800-526-3593 or www.biotech.pharmacia.se)
DocuGel Workstation, BenchTop I Scanner, and BenchTop II Scanning Densitometer
The DocuGel Gel Documentation Workstation is fully integrated, featuring a CCD with zoom lens, an optional Sony 256 gray scale thermal printer, and can operate from a PC or Macintosh platform. The BenchTop I is a 12-bit scanner with an optical density (O.D.) range of 0 to 2.4, while the BenchTop II is also a 12-bit scanner with an O.D. range of 0 to 3.0. Both are compatible with any printer with a Windows driver and operate from a PC platform. The BenchTop I can handle 8.5 x 14" color or black and white images, while the BenchTop II can accept large format items, up to 14 x 17". (978-663-8161 or www.scanalytics.com)
Eagle Eye II Still Video System
Optimized for chemiluminescence, the Eagle Eye II is a versatile system comprised of a light-tight cabinet, customized, high-resolution CCD, zoom lens, printer, Pentium computer, and color monitor. The Eagle Eye II can acquire almost any type of data, from ethidium bromide gels, to Syber¨-, Sypro¨-, and silver-stained gels, to luminescent microplate assays, and even thin layer chromatography plates. Numerous hardware options are available, too. For example, users can select their own PC and a variety of printers are compatible with the Eagle Eye II. EagleSight Acquisition and Analysis Software is Windows-based, has seemingly limitless capabilities, and free upgrades are available for the life of the instrument. New features of EagleSight software include time-lapse photography, colony counting, molecular weight and band intensity measurement, and image superimposition. Data can be saved in several formats or exported to spreadsheet programs, slide making programs, or word processing applications. Integration occurs at the chip level, and the Eagle Eye II supports additional hardware peripherals. (800-424-5444 or www.stratagene.com)
Ultra-Imager-5000, Big-Gel-Imager-4500, and KS-4000-Slide-Imager
Ultra-Lum's light-tight systems include patent pending Ultra-GloTM UV-to-Visible Light Converter, a high-resolution integrating CCD camera, motorized zoom lens, 256 gray scale thermal printer, PCI frame grabber, and Image-PC software, and can integrate into any existing Pentium or PowerMac computer. The Ultra-Imager-5000 features a small, light tight darkroom cabinet. The KS-4000-Slide-Imager has a built-in camera slide for viewing gels/blots up to 20 x 20 cm, while the Big-Gel-Imager-4500 is ideal for viewing gels up to 20 x 40 cm in a benchtop darkroom cabinet. Combined with Ultra-Lum's patented Electronic Transilluminators, Ultra-GloTM UV/Visible Light Converter allows for up to three wavelengths plus visible light under any Ultra-Lum benchtop darkroom by adding visible transillumination. (562-529-5959 or www.ultralum.com)
GDS-8000, 7600, 7500, and PhotoDOC-It Systems
The GDS-8000 is a PC-based system allowing image acquisition and analysis at the same location. It features a choice of computer as well as PCI image acquisition boards to trigger a variety of cameras, including cooled or non-cooled CCDs or three-chip color CCDs for microscopy. The GDS-7600 is similar to the GDS-8000, but is a Macintosh-based system. The GDS-7500 comes with an ImageStoreTM processing unit, complete with a video capture card and software, 3-1/2" floppy drive, RS232 SCSI port, and mouse. The GDS-8000, 7600, and 7500 are available with a choice of UVP darkroom and transilluminator. The PhotoDOC-ITTM System is a compact imaging workstation featuring a non-cooled CCD with zoom lens, monochrome monitor, darkroom, transilluminator, and DOC-ITTM Link processing unit allowing extended camera integration. UVP also offers accurate and flexible image analysis software. (800-452-6788 or www.uvp.com)
VILBER LOURMAT Photo-Print System
At the core of the Photo-Print System is a CCD with built-in zoom lens with interference filter, allowing photography of gels ranging from 2 to 25 cm in length with no lens change. Photo-Print's front panel provides simple selection of real-time viewing, integration time presets, and image freezing. A capture option enables saving images on a PC running under Windows 3.1 or 95. Designed for use with VILBER LOURMAT transilluminators, the compact Photo-Print System is ideal for labs where space is at a premium. More information can be obtained by faxing the French Technology Press Office, Inc. (312-222-1237) or faxing VILBER LOURMAT (33-1-64-80-48-59)
Thomas Unger is Vice President of Research and Development for Miragen Inc. The authors can be reached at email@example.com.