More Than Just A Pretty Picture

to keep up with ever expanding applications. Date: October 26, 1998Comparison of Products Ten years ago, a "gel documentation" system meant an instant camera on a pole over a transilluminator. As charged-couple device (CCD) cameras became less expensive, electronic hardware to create digital images became available, and sophisticated image enhancement and analysis software was developed, electronic gel documentation systems were introduced and have become an essential laboratory instrument for molecular biological research. Researchers can choose among a large variety of systems depending on their needs and budget. With so many choices, it can be difficult for a first-time buyer to select the best system for his or her needs. SYBR® Green I Stained Double Stranded DNA in Acrylamide Gels detected with Hitachi's FM-BIO. Before investigating the plethora of systems and the variety of options available, it is important to have a clear idea of your needs. Amy Edwards, technical product support for Ultra-Lum, alludes to this when she advises researchers to know what they want before they shop around. According to Edwards, Ultra-Lum prides itself on modularity and will customize a system to the needs of the customer. In fact, this is true for most companies, so it is very important to know what you need. Who will be using the system is one of the most important initial considerations. Brian Walsh, vice president of Fotodyne, says that the first thing he asks is, "will the system in the lab be dedicated to only one or a few people?" He mentioned that one system often serves several labs in a department. Christine Ethier, product manager at Genomic Solutions, also felt that this was an important consideration and explained, "For a multiuser situation, you want a system that saves the image file to a floppy that can be transferred to another computer for analysis. This avoids a bottleneck." Conversely, in a more dedicated situation with only one or a few users, a single system that has extensive analysis capability may be more suitable. Next, it is useful to assess the current resources available. Aside from the image collection device (i.e., camera or scanner) and the hardware, gel documentation systems contain many additional components that may or may not be required, depending on what the lab currently has available. For example, if a large, spacious darkroom is readily accessible, a benchtop cabinet darkroom may not be necessary, or if the lab currently has three transilluminators, another might just add cost to the system. Another concern is that some systems are better for certain applications than others. Dale Richardson, senior product manager for software and gel documentation systems at Bio-Rad, mentioned that he instructs the sales force to first find out, "What is the customer trying to accomplish? Will the system be just for basic gel recording? If gel analysis will be involved, what type and how complex?" It is important to determine both the type of data that will be collected and the type of analysis required. Is it primarily to collect data from sequencing gels, document images on x-ray film, record bands on agarose gels, look at coomassie-stained Westerns, or visualize chemiluminescence results. Also, will it be important to accurately quantify the amount of sample in a specific band, the precise size or weight of the sample, etc.? Also, having an idea of common types of problems or difficulties encountered with the data is helpful. Are there often doublets, faint bands, or a lot of background? Analysis Window of Microbiology International's GeneGenius, showing lanes on tracks, band profiles, molecular weight calibration curve and results table. With respect to applications, there are really two aspects to think about. The first is to ensure that the hardware is capable of collecting the appropriate data. The second, and more difficult aspect to assess is to ensure that the software produces reliable results. For example, Richardson mentioned DNA fingerprinting analysis as an example where sensitivity and density measurements are not important for quantitative results, whereas for other applications, accurate values for these parameters might be essential. The ability of various algorithms to generate accurate and reproducible results for the different parameters varies significantly from system to system. It is also useful to consider other imaging needs in addition to gel documentation. Nicole Berry, a marketing business manager for Kodak's Scientific Imaging Systems group notes, "If a lab's imaging needs go beyond gel documentation, look for a system that provides flexibility in addition to high performance." In this context, she was referring to the Kodak Digital Science EDAS system with it's detachable megapixel camera that can be mounted on a microscope or used for "point and shoot" photography in the lab or in the field. However, certain labs may have other related applications that can be addressed by a particular system. Specifically, several dual imaging systems are on the market (see table) that provide platforms for color microscopy imaging, as well as gel documentation. A principal component of any gel documentation system is the image collection device. The market is dominated by camera-based systems, most of which use CCD cameras. In a CCD camera, images are created when photons entering the camera lens activate CCDs arrayed on a half inch sensor chip behind the lens. A digital image is recorded by converting the activated CCDs to pixels--the more pixels, the better the resolution. The minimum standard is 640 x 480 pixels. However, the full level of resolution is achieved only if the gel image completely fills the sensor. To accomplish this for various size gels requires a lens with an adjustable zoom feature. If the lens cannot be adjusted to make a gel fill the frame, resolution will be lost. Dual layer micrograph of bovine pulmonary artery endothelial cells; Red labeled mitochondria, green labeled F-actin filaments captured with the Kodak Digital Science MDS 120 system. One key design factor that determines camera performance is the digitization format. The CCD produces analogue data (electrons) that must be converted to digital data before the software can analyze it. Less expensive cameras provide 8-bit data. More expensive cameras provide 12-bit and 16-bit data. The 12 and 16-bit cameras have the advantage of being able to quantitate data over a broader dynamic range. A 16-bit camera can accurately measure both strong and weak signals from the same blot or gel, while an 8-bit camera will have difficulty doing this. However, high dynamic range can come at a cost, both with respect to noise and transfer time. Another feature to look at is the lux rating. The lower the lux rating, the fewer photons it takes to activate a CCD, the more sensitive the camera. Also, over a period of time, energy in the form of heat can activate pixels, creating background, known as thermal noise. This is an issue particularly for chemiluminescent applications since they require longer exposure times. To minimize this problem for these applications, a cooled CCD camera is recommended. In addition, for long exposures, Doug Drake, product manager at Stratagene, notes that the output amplifier circuitry--the unit that transfers and amplifies the signal from the CCD array to the frame-grabber board--can often introduce more background noise than thermal activation of the CCDs. Deborah Smead, product manager for Hitachi Genetic Systems, noted, "Gel documentation is really a broader term that has been adopted by the manufacturers of the camera-based systems." Several systems on the market use a scanner rather than a camera for image collection. Standard scanner-based systems have a broader dynamic range, provide better resolution than 8-bit CCD cameras, and are more economical than comparable camera-based systems. However, standard scanners are limited to imaging gels stained with colorimetric dyes or autorads, so they are not suitable for DNA gels. For fluorescent imaging--as would be necessary to visualize fluorescently labeled DNA--a more advanced laser scanner, such as Hitachi's FMBIO II or Molecular Dynamics' Storm system, is required. Although such systems offer several advantages over the camera-based systems for some applications, they are two to three times more expensive than the basic camera-based systems. Additional information on cameras and scanners can be found in "Picture Perfect-Systems for Gel Documentation and Analysis" in the December 8, 1997, issue of The Scientist. Roche Molecular Biochemical's Lumi-Imager, showing the ability to analyze two color fluorescence. Aside from the image collection device, the software is probably the most important element of a system. There is considerable variability in the software across the market. Some systems can be purchased with complete analysis software; others simply digitize the data and save it to a disk. The image can then be transferred to a separate computer and analyzed using any number of analysis programs. It is important to carefully evaluate any software to insure it provides the quality of results desired. Len Hook, product manager at Scanalytics, Inc., feels that the software is really what differentiates various gel documentation packages. He suggests looking at the sophistication of the background subtraction and gaussian deconvolution of overlapping bands. Jim Bower, director of operations at MedRx International Technologies, also expressed concern about the ability of some software to handle sophisticated analysis and recommended that researchers make sure the software can deal with background. He mentioned that it is "difficult to get rid of background and not also get rid of some of the sample" and suggested testing the software with a known quantity of sample on a variable background. Doug Kelley, director of sales for UVP, suggested testing the reproducibility of the software by having it recalculate various measurements (for example, molecular weight, density, mass loading) several times from the same image. He mentioned that some programs can give considerable variance due to inaccurate algorithms. Also, Kelley noted, look for a company that supplies a completely integrated system and not just individual components. This will increase the reliability of the system and minimize future problems. Brian Holaway, product manager at Roche Molecular Biochemicals (Boehringer Mannheim), noted that many systems "do not provide uniform measurements which are independent of sample position." To test the flatfield capability of a particular instrument, he suggested researchers take readings at all four corners and in the center of the imaging field. The readings from the sample should be the same, independent of sample position. In addition to insuring the software is capable of performing the analysis needed, a system must be somewhat user-friendly. To assess this quality, it is important to look at the steps required to access the quantitative and computational capabilities. Chuck Parzick, president of Digital Imagers, felt ease-of-use was the most important consideration when purchasing a gel documentation system. He mentioned that the Digital Imaging system requires only three mouse clicks to take the image and an additional three to print it. Ethier emphasized the importance of an easy-to-use system, especially in a multiuser environment where there is a significant turnover of researchers. He noted that researchers "don't want to have to learn an instrument but just want to use it as a tool for research." The way images are saved can also be critical, especially for stand-alone systems where the image is transferred to another computer for analysis. Some systems allow you to save data in a number of graphic formats (e.g., GIF, PCX, BMP, TIFF, and JPEG). It is important to consider compatibility if you will be porting the images to different computers. Another factor affected by the format is the amount of memory required to store an image. For instance, Bower notes that a JPEG file is about one eighth the size of a TIFF file containing the same image, although for some quantitation, JPEG files may not be appropriate because data may be lost. Prove it for Yourself A final general consideration involves logistics. How easy will it be to get the gels in and out of the darkroom cabinet? If you run many very small or large gels, will it be convenient to document them with the system? Often these needs can be addressed by requesting that different components be substituted for those that are offered as standard parts of the system. Imaged by Alpha Innotech's Spectra Image™ 5000 A company should be willing to provide an in-house demonstration of any system you are considering. James Lee, product manager at Alpha Innotech Corporation pointed out that paper specs do not always determine the best performing system for a customer. Lee adds, "During the in house demos the customer should be focusing on speed"--how quickly one can go from image to analysis--"ease of use--how easy it is to perform complex task--and versatility--how many tasks can be performed by the imaging station."In addition, with so many choices available, it is likely that you might want to see several systems demonstrated. Guenter Dietzel, president of Raytest, suggested that researchers invite several companies onsite at the same time to demonstrate of their instruments with the same sample. In addition to enabling researchers to compare several systems efficiently, this type of comparison also rules out differences in the chemistry from sample to sample. As with any technology, today's cutting-edge developments are tomorrow's old news. When shopping around, it helps to keep in mind where the technology is going. Sia Ghazvini, vice president of business development at Nucleo Tech Corporation, specifically cautions researchers not to back themselves into a corner by purchasing a system without expansion options. He suggests that researchers, "look for a company that provides ongoing hardware and software development to support their systems." Chad Miller sees documentation systems offering more complete solutions for an expanding number of applications. As an example, he mentioned a recent collaboration between Molecular Dynamic and CLONTECH that focused on developing approaches to analyze data generated using membrane-based cDNA arrays. Miller believes new applications will direct the product development of imaging and documentation systems. Ghazvini of Nucleo Tech Corporation agrees with Miller and already considers the term "gel documentation" a misnomer since many of today's systems provide image processing and analysis functions that surpass simple documentation. He pointed out that current systems can actually get more information out of a gel than is possible without a system." He mentioned, for example, that edge-finding algorithms in many analysis packages can often bring out faint bands present--but not visible by eyeþin a smeared PCR reaction on a gel. He sees the role of these systems continually expanding and offering increased ability to analyze, quantify, and organize data and information from a wider and wider range of applications. Comparison of Products Author Paul Diehl, can be reached at paul_vfocus@msn.com

More Than Just A Pretty Picture

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