Exposing Gel Documentation Systems

Remarkable advances in gel documentation technology in the last 15 years have made the quantification and identification of protein and DNA faster, easier, and more readily available for a broad range of analyses. Fuzzy bands, precious single-copy pictures, and eyeballed judgments are quickly becoming things of the past, replaced by digital imaging and fast computer analysis. The latest imaging systems can accommodate a variety of gels and staining techniques while accurately and precisely recording, optimizing, quantifying, and storing the data. The most advanced systems, which were previously reviewed in The Scientist,1 can be priced in the $70,000 neighborhood. But for researchers facing more modest budgets or less-complex needs, there are plenty of more basic and affordable systems, ranging in price from $4,000 to $10,000. In fact, according to John Fox, president of Encinitas, Calif.-based Lightools Research, it is a buyer's market. Basic gel documentation systems "have become a commodity product. [Companies] compete on the basis of price rather than performance," he notes. They also compete by providing products that meet as broad a range of needs as possible. In other words, there is an affordable system to meet the needs of nearly every lab. There are five basic components to a complete gel documentation system: an imaging device, a light source, a computer, analysis software, and a printer. A number of companies allow cost-conscious buyers to custom configure systems to avoid purchasing components they may already have, or that are unnecessary. Amy Bohn, sales coordinator for Fotodyne of Hartland, Wis., notes that because every lab has a different set of requirements, there is demand for "build-your-own imaging systems." Fotodyne offers this type of versatility with its new Express system, allowing researchers to pick and choose among an assortment of transilluminators, cameras, and imaging software. The FOTO/Analyst Express is specifically designed to accommodate any kind of transillumination and is therefore well suited for a laboratory with diverse needs. Frederick, Md.-based Syngene offers two modular product lines. The company's new DigiGenius line includes a four/eight-megapixel digital camera that can be attached to a compact hood or mini-darkroom to accommodate bench space limitations. Zoom and focus modes allow the user to capture images directly into the company's GeneTools analysis software; no additional hardware or separate acquisition software is needed. Syngene's GeneCam Flexi line is also highly modular, allowing users to customize nearly every component. As with the DigiGenius, the GeneCam Flexi line of products allows researchers to choose the camera lens type (fixed or zoom), a camera hood, camera stand, or mini-darkroom. Transilluminators and thermal printers are also optional components, and the total system price depends on the final configuration the user chooses. Syngene provides the integrating GeneSnap and GeneTools software with all systems. Ready... Aim... Fire! Gel documentation technology has come a long way since the days scientists used instant cameras mounted above a UV box. There is now a virtual arsenal of imaging systems, most of which operate with either a CCD (charge coupled device) camera or flatbed scanner. Both types have advantages as well as drawbacks, each of which must be carefully considered prior to selecting a system. The sensitivity of CCD cameras has dramatically affected the way scientists record and analyze data (reviewed in a previous issue of The Scientist).2 The high resolution and signal intensity of these cameras make them more suitable than film-based systems for accurately recording data. In addition, as CCD cameras can print to thermal printers and allow image viewing in real time, researchers can fine-tune captured images. In contrast, getting a high-quality image from a traditional film-based gel documentation system is usually a laborious process that involves several exposures on expensive film. At the heart of a CCD camera is an array of light-sensitive "picture elements" (pixels) that records the intensity of incoming light. By processing this array of intensities, a computer can reconstruct the original image. In low-end CCD cameras, the exposure time is 1/30 of a second. This short exposure time is adequate for imaging protein gels and autoradiographs but not for most DNA gels, which must be viewed with a relatively dim UV light. A feature called integration allows the CCD to compensate for these low light levels and obtain a timed exposure. The highest-end cameras allow users to integrate for a specific length of time, or until the image reaches the desired brightness (dynamic integration). Integration requires two hardware components that can dramatically increase the price of a system: an integrating camera and a frame grabber to convert the camera's analog signal into a digital image. Several properties of CCD cameras can affect image quality and the ability to perform more difficult operations. The bit depth refers to the number of bits used to represent the brightness of a particular spot on an image. John Bradshaw, product manager for Alpha Innotech of San Leandro, Calif., notes that although many researchers assume higher bit depths will mean better data, "in reality, the actual dynamic range provided by eight bits is just fine for most gel documentation applications." Eight-bit cameras, which yield 256 levels of gray-scale brightness, are sufficient for most uses.3 Other cameras come equipped with 10-, 12-, or 16-bit gray scales and offer a greater potential dynamic range, which may be required for protocols such as chemiluminescence. Most frame grabbers digitize to an eight-bit image. The camera's pixel concentration largely determines its resolution, or the number of picture elements into which the camera dissects the image for storage and transmission, whereas the sensitivity, measured in lux, determines the minimum amount of light a camera can detect. Sensitivity is important for protocols involving low-level emission light. With these lower-light protocols, however, comes the accompanying problem of background interference. According to Chuck Parzick, president of Digital Imagers of Cathedral City, Calif., the lack of adequate filtration can hamper data analysis by allowing faint bands to disappear while strong bands bloom into overlap. A weakly filtered camera can also detect source light concentrations and scattered rays, both of which compromise light emission from the bands. To address these problems, Digital Imagers offers bandpass and polarization filters on its Gel QuickPrint™ and BioVideo-500™ systems. Bandpass filters allow only the narrow window of light that includes the emission from the sample to pass, whereas polarization filters deflect incidental light, allowing only the perpendicular light to pass. For example, a user imaging a gel stained with ethidium bromide would only want to see light around 580 nm. A 580 +/- 20 nm bandpass filter will help create a uniform, black background that appears very flat in the detection and analysis phases. A polarization filter allows the user to see weak bands on the same image as strong ones and doublets as two bands instead of one. High-end cameras are often cooled to reduce thermal noise that builds up as the image develops. Researchers who work with gels that often produce faint or doubled bands may want to consider a cooled camera system, though they are usually considerably more expensive than other systems detailed in this profile.4 Some gel documentation systems include a digital camera instead of a CCD camera. There are a wide variety of digital cameras available, but not all are suitable for biological applications. Unlike CCD cameras, digital cameras do not support real-time video imaging. However, they offer excellent image quality. For example, the pictures produced by the KODAK DC290 camera, the main component of Rochester, N.Y.-based Eastman Kodak's EDAS 290 digital camera system, are up to six times sharper than comparably priced CCD cameras. The camera is also highly sensitive, being able to detect as little as 30 pg of ethidium bromide or SYBR green-stained DNA in an agarose gel. The EDAS 290 system includes KODAK 1D Image Analysis Software version 3.5. In September, Eastman Kodak introduced a new version of the EDAS 290 system that includes optional epi-illumination built into the imaging cabinet. As an alternative to either digital or CCD cameras, some systems come bundled with scanners. Traditionally, researchers have used these devices to analyze autoradiographs or blots. Recent developments now allow investigators to use scanners to image gels stained with dyes such as Coomassie blue, which do not require transillumination. However, scanners still cannot typically be used for UV-based imaging of ethidium bromide-stained gels, for example. Since scanners provide a greater uniformity of illumination and have an advantage in resolution, they often provide more reliable density analysis than CCD cameras.5 The market for scanners, and therefore their availability, is somewhat more limited than that for CCD camera-based systems. However, Amersham Pharmacia Biotech of Piscataway, N.J., offers an affordable ImageScanner that can acquire any kind of visual stain with high sensitivity and can be set by the user to operate in transmission mode (for gels) or in blot mode (for thin layer chromatograms or autoradiographs). Light 'Em Up Even the most sophisticated camera requires an adequate light source. Transilluminators and epi-illuminators provide the necessary light to produce high-quality images. Transillumination is suitable for transparent subjects such as gels because it sends light through the sample toward the camera. Epi-illuminators provide a light source on the same side of the sample as the camera and are more suitable for imaging blots. Since most transilluminators use fluorescent lamps similar to the omnipresent laboratory overhead light, they sometimes produce a slight flicker that may cause a "beat" frequency to appear in the picture at low light levels. Ultra-Lum of Claremont, Calif., has overcome this flicker by patenting a high-frequency electronic circuit that runs at approximately 25,000 cycles per second (Hz) (as opposed to the standard 60 Hz) so that the flicker is beyond the scanning rate of any camera and therefore does not affect the quality of the picture. This technology produces flicker- free viewing and images free of beat frequency artifacts, and it has the added advantage of extending the life of the lamp. Ultra-Lum also offers infinite-variable intensity electronic transilluminators that allow users to adjust the UV intensity from 0 to 100 percent during gel documentation. Thus, a researcher can excise a band of interest under low intensity UV light to minimize photo-nicking, and then raise the intensity to whatever level is needed for maximum fluorescence for photography. Another factor to consider is whether both UV and white lights will be necessary. Because UV light will not penetrate ordinary glass, manufacturers frequently include two glass surfaces in their light box cabinets that provide both kinds of light, or provide a converter plate that changes UV light into white. White light is often not required and in some cases can be replaced with other wavelengths. For example, Ultra-Lum's Ultra-Glo™ UV-to-visible converter plate converts UV into red or orange light to provide higher contrast and better readability of Coomassie blue-stained gels. If darkroom space is limited, a light-tight cabinet that encloses the box may be a worthwhile investment. Anthony Alburo, a technical support specialist for Bio-Rad of Hercules, Calif., notes that the biggest advantage of the company's GelDoc 2000 package over its competitor is this feature. "We have the tightest light-tight hood on the market. With it, background and ambient light are significantly reduced," he notes. Damage Assessment In today's competitive market, many gel documentation systems come equipped with analysis software, even at the low end of the scale. A typical package directed at 1-D analysis is able to define lanes and mark bands, allowing the user to view profiles of the lanes either individually or overlaid for comparison. Frequently, the software determines basic molecular weight information and sometimes the quantities at each molecular weight. More sophisticated packages, which if sold separately can cost more than $5,000, offer specialized tools for restriction fragment length polymorphism (RFLP), gene sequencing, and 2-D analysis. Public domain shareware such as the NIH Image and Scion Image software used by Digital Imagers can be downloaded and updated free of charge. Both are designed for capture, enhancement, archiving, annotation, printing, and general gel analysis. Macro programming capabilities allow the user to create specialized functions. On the other hand, Alpha Innotech designs its proprietary software to provide the highest level of user friendliness. "We are an applications solution company," product manager Bradshaw explains, adding: "For basic gel documentation, the most important thing is convenience." The company's Ease of Use™ software, which is provided with the AlphaImager and AlphaDigidoc gel documentation systems, "allows the user to capture and document an image in just a couple of clicks," Bradshaw says. Many companies include thermal printers in their gel documentation packages, but 600-dpi laser printers will often suffice and may save money for the lab as well. To obtain publication-quality prints, users can bring their disks to a local printing company, while using laser printouts for personal use. The image will be conserved on disk for future use and analysis. A final consideration is "traffic management." If there are going to be a number of users, consider a system that not only provides a solid image but also keeps the queue moving. According to Lightools Research's John Fox, busy labs would be best outfitted with a system that has "a centralized gel documentation and data collection unit with offline analysis to prevent the usual bottleneck" that occurs while researchers wait for a previous user to analyze his or her data at the documentation site. Before making a purchase, consider that most of the work with gel documentation systems occurs on the computer that drives the camera. Therefore, the most useful system will be one that can be easily and intuitively controlled with the analysis software, without physically adjusting the camera settings. Some important software features to keep in mind when comparing gel documentation systems are image capture (zoom, pan, and exposure control), image processing (brightness, contrast, and flip), annotation, analysis (background correction, quantitation, and comparison graphs), and archiving. Most companies will provide an in-house demonstration on request. And chances are, they'll arrive with plenty of ammunition. Sarah Goforth (sarahgoforth@hotmail.com) is a freelance writer in Madison, Wis. References 1. P. Diehl, "Know yourself first," The Scientist 12[21]:19, Oct. 26, 1998. 2. G. Meisenholder, "Reality bytes," The Scientist 13[17]:20, Aug. 30, 1999. 3. M. Miller et al., "Digital imaging considerations for gel electro- phoresis analysis systems," Electrophoresis, 22:791-800, March 2001. 4. L. DeFrancesco, "Illuminating images," The Scientist 12[4]:16, Feb. 16, 1998. 5. K. Muira et al., "Imaging and detection technologies for image analysis in electrophoresis," Electrophoresis, 22: 801-13, March 2001. Supplemental Table Click to view the PDF file: Gel Documentation Systems Under $10,000

Exposing Gel Documentation Systems

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