Advancing with Gel Documentation Systems

Courtesy of UVItec UVItec's UVIscan system Most life scientists own lab notebooks bursting with Polaroid film, each frame capturing a piece of agarose-embedded information. Whether those images are priceless or mundane, crystal-clear or blurry, all are one-of-a-kind: Spill a cup of coffee on the notebook, and a piece of data could be lost forever. But thanks to digital gel documentation systems, such worries are receding. "Basic documentation systems have become commodities in the laboratory, part of people's startup packages and maintenance packages," says Renee LeMaire-Adkins, a product line manager at Bio-Rad Laboratories, Hercules, Calif. A gel documentation system consists fundamentally of a camera, a light source, and a medium for recording images. Traditionally, instant-film cameras satisfied these requirements, and the simplest digital systems--the SpeedLight Jr., from Lightools Research in Encinito, Calif., and the Printgraph manufactured by Atto of Bunkyo-Ku, Japan, for instance--deliver similar functionality, substituting a video or digital camera and a thermal or standard PC printer. But modern gel documentation systems can also save images electronically. "The primary push is collecting digital data," notes LeMaire-Adkins. "People are tired of having photographs floating around. Also, digital data allows good quantitation and additional analysis with [a computer and] appropriate software." Advanced imaging systems can still cost as much as $70,000 (US),1 but the functionality of systems costing less than $20,000 is often sufficient for individual labs and enables imaging of a variety of fluorescent, visual, and sometimes even chemiluminescent samples.2 Courtesy of Syngene The DigiGenius digital camera with hood and optional transilluminator. THE BASICS ON CCD The "film" in most digital gel documentation systems is actually a charge-coupled device (CCD). This tiny, light-sensitive semiconductor stores a charge in proportion to the number of incoming photons it encounters.3 These devices are fabricated in arrays on an insulated background, an assembly referred to as a CCD chip. After exposure to a light source, CCD chips are "read out" serially: The charge in each CCD element, or pixel, is converted to a voltage reflecting the amount of light it received. A conventional CCD camera outputs its analog voltage signal either directly to a monitor or printer, or to a frame grabber, which is an analog-to-digital converter (ADC) that allows a computer to interpret the CCD signal. Low-end gel documentation systems, however, increasingly employ digital cameras, which have built-in ADCs. CAMERA CONSIDERATIONS All CCDs are not created equal; variables include bit depth, resolution, and noise- reduction capabilities. The amount of charge each CCD element is capable of storing determines its bit depth--the number of bits needed to describe the possible states of the CCD without losing information. An eight-bit CCD, for instance, can store 28 or 256 shades of gray, while a 16-bit CCD can hold 65,536. Conventional CCD cameras record monochrome images, whereas most digital cameras record eight-bit images from sets of pixels sensitive to red, green, and blue light. Since gel information is typically monochrome, digital images are often converted to eight-bit grayscale before analysis. Image intensity, exposure (or integration) time, and bit depth are related variables. Longer integration times are required to visualize faint signals, but as integration time increases, thermal noise--or dark current--accumulates in the CCD. As a result, fewer states above the background noise level exist to distinguish a signal. A higher-bit-depth CCD fills up with dark current more slowly, allowing better visualization of faint signals. Alternatively, cooling the CCD chip, typically using an attached Peltier module, can reduce background noise. "All electrical currents have noise," explains John Bradshaw, product manager at Alpha Innotech in San Leandro, Calif. "By putting a little refrigerator right on the chip you substantially reduce your dark noise." These cooled video CCD chips are up to four times as sensitive as noncooled ones, says Len Hook, senior scientist with Fairfax, Va.-based Scanalytics. Another important specification is resolution, but higher resolution does not necessarily imply better gel documentation. The EDAS 290 system, from Eastman Kodak Company's Scientific Imaging Systems of Rochester, NY, for instance, includes a digital camera with a 2.1-megapixel CCD recording element. According to Liz White, product manager, this camera features a higher signal-to-noise ratio and better sensitivity than many cameras with higher resolution. "The more pixels you have on a CCD of the same size, the smaller the pixel. The smaller the pixel, the less signal the pixel can hold, resulting in a lower signal-to-noise ratio [and lower sensitivity]. Number of pixels is much less important for gel documentation, because the bands are relatively large." High-end CCD chips can be extremely expensive. But Hook says, "If a lab is only involved in traditional fluorescent gels--SyBr Green, ethidium bromide, and so on--a low-end camera [noncooled eight-bit] is generally sufficient." Such a camera will also suffice to visualize gels stained with dyes such as Coomassie blue. Maximum integration time increases with these low-end chips, from about one second to five seconds, "but typically that is more than enough to see even a faint fluorescent band," says Hook. More expensive equipment is typically required for chemiluminescence detection, however, because such applications usually require much longer integration times. Courtesy of Scanalytics BETTER THAN FILM: Digital gel documentation systems are supplanting traditional film for data archiving and analysis. This screenshot underscores why: shown are molecular weight analysis and band quantitation, as well as correction for band "smiling" and partial resolution. DARKNESS AND LIGHT There are three basic types of darkroom hardware--dedicated darkroom mounts, portable hoods, and cabinet-style enclosures. Dedicated darkroom mounts and portable hoods are often cheaper and are easy to use with existing darkrooms and transilluminators. Cabinet-style enclosures tend to be more expensive, but can be used as stand-alone imaging stations without a separate, dedicated darkroom. Lighting hardware comes in two major varieties, categorized by the side of the sample on which the light originates. Transilluminators transmit light through the sample to the camera, and are well-suited for gels and similarly transparent items. Ultraviolet transilluminators, used for imaging fluorescent stains, are the most common type, but many manufacturers also offer white-light transilluminators, or conversion screens that transform the output of a UV transilluminator to white light. Epi-illumination supplies light to the sample from the same side as the camera by reflection, and is better for opaque samples such as blots. Epi-illumination sources must be mounted above the sample, and so are more compatible with cabinet-style darkrooms. White-light epi-illumination is a common option, but users requiring epi-UV or specialized lighting should consult the manufacturer to make sure an appropriate source is available. Light-filtering devices, particularly important for fluorescent protocols, vary considerably between systems. Most manufacturers include filters for visualizing ethidium bromide-stained DNA, but price, availability, and inclusion of other filters varies widely. Courtesy of Syngene Syngene's GeneGenius system SOFTWARE OPTIONS After imaging hardware, the most important purchasing consideration is image analysis software. The features and usability of these applications vary, but minimally, gel analysis software should be able to detect lanes and bands and include tools for gel annotation. Molecular weight determination and product-yield calculation are also fundamental, and are provided in many packages. The more full-featured software packages offer audit trails for compliance with good laboratory practice, as well as additional gel analysis tools, and extra features such as colony counting. Because of this variability, it is particularly important to try before you buy, especially if you anticipate the need for frequent or complex image analyses. Labs with substantial gel analysis loads will most likely benefit from the sophisticated commercial software and hardware packages available. Users who do not require sensitive quantitative image analysis, however, can switch from hardcopy to digital documentation with little investment or fuss. Two free software packages, NIH Image and ImageJ (rsb.info.nih.gov), are widely used for this purpose. James Burnette, a postdoctoral fellow at the University of Virginia in Charlottesville, has even written instructions for building such a system (www.people.virginia.edu/~jb6vz/gel). "Almost any digital camera will work well for imaging gels if it has a zoom feature, can control the aperture, and can be used with appropriate filters," he says. One item to consider in setting up a gel documentation system is its likely traffic flow. Having multiple copies of the imaging analysis software allows users to acquire images at the gel documentation station and analyze them at their own PCs, thereby relieving crowding and bad tempers. Though users of free software can install as many copies of their analysis software as they want, users of commercial products must consider licensing issues. Several manufacturers have addressed this problem. Bio-Rad provides users with multiple copies of a "basic" version of its software, and Frederick, Md.-based Syngene provides two copies of GeneTools analysis software with each purchased system. Eastman Kodak takes an alternative approach, says Sharon Goodkowsky, marketing manager for gel documentation systems. The company's 1D Image Analysis Software has "optional concurrent user licensing," allowing network administrators to install the software on all computers and centrally control the number of simultaneous users. Courtesy of Polaroid Polaroid's portable hood-based DigitalGelCam system. AS YOU LIKE IT While many systems support a wide range of visual and fluorescent imaging applications, not all do. The GeneGnome system from Syngene, for example, is a specialized chemiluminescence-only system. Scanner-based systems that address only visual gel documentation include: Fullerton, Calif.-based Advanced American Biotechnology's TR-500S Imaging Densitometer; Syngene's GeneScan; Piscataway, NJ-based Amersham Biosciences' Image-Scanner; and Cambridge, UK-based UVItec's UVIScan. The major differences between these scanner-based products and cheaper, commercially available scanners with similar specifications are bundled software and transparency-scanning capability. The latter is unnecessary for qualitative gel archives, but crucial for accurate densitometry. "You can get perfectly good images of gels using reflective scanning, but they are not quantitative," says UVItec's Sales and Marketing Director, Paul Goulding. "The light source is going through the gel, bouncing off the white background and going back through the gel to get the image." The central theme voiced by many gel documentation manufacturers is customizability. A wide variety of camera and lighting specifications can be built into a given cabinet or dedicated darkroom-mounted system, and researchers must ultimately decide how to personalize a system to suit their needs. But be advised: options such as thermal printers and computer systems can add thousands to the final system price. Labs willing to undertake setup of computer hardware and software may save a considerable sum by carefully considering their needs. A quick glance at minimum hardware specifications for analysis software packages, for instance, suggests that even labs with substantial image analysis needs are unlikely to require the latest model computer hardware. Similar issues apply to printer choice. Despite the lure of paperless documentation and analysis, many researchers prefer to have hard copy of a gel image for backup or journal submission. For this purpose, thermal printers, which generate a photographic-quality, continuous-tone image, are generally preferred. High resolution laser and inkjet printers, however, produce near-photographic quality images suitable for personal use and archiving, and as journals increasingly encourage electronic submissions, a dedicated thermal printer is often more an option than a requirement. Jeremy Pierce (jlpeirce@Princeton.edu) is a freelance writer in Philadelphia.

Advancing with Gel Documentation Systems

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