Image acquisition and analysis

Adapted from Chapter 7, Gel Electrophoresis of Proteins, by David E. Garfin, Pages 197-268, in Essential Cell Biology, Volume 1: Cell Structure, A Practical Approach, Edited by John Davey and Mike Lord, Oxford University Press, Oxford UK (2003). Used by permission of Oxford University Press.

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Figure 1. Image analysis software for 1-D and 2-D gels. (A) A 1-D analysis software window is shown. Densitometric analyses of the column and row identified by the crosshairs appear above and at the left of the gel image. (B) In this 2-D software window, bar-graph annotations are shown representing the intensity patterns of selected protein spots in several related gels. This type of analysis is useful in comparing differing test samples of proteins.

Once gels have been stained, it is often sufficient to examine them visually. The requirements of some types of experiments are satisfied by simple comparisons of the band patterns in relevant lanes in gels. In other situations, migration distances of selected proteins can be measured by hand with a ruler and standard curves drawn on graph paper. Simple analyses like these are usually done on the wet gels. Sometimes it is advantageous to photograph a stained gel and use the photographic image for measurements. In regards to archiving gel information, it is easier to tape photographs of gel images to notebook pages than to store wet gels or gels dried on cellophane or filter paper.

In laboratories where large numbers of gels are run on a routine basis, digital methods for image acquisition and data analysis have replaced wet gels and photographs (1, 2). Several types of imaging systems and associated software are commercially available for analyzing gels stained with just about any kind of stain. These instruments greatly simplify data acquisition and analysis and the archiving of gel patterns.

The three categories of image-acquisition devices used in with electrophoresis gels are (i) document scanners, (ii) charge-coupled device (CCD) cameras, and (iii) laser- based detectors. Document scanners as configured for densitometry are for measurements on gels stained with one of the colored materials, CBB or silver. They operate in visible light illumination, 400-750 nm, with dynamic ranges extending to 3 O.D. The linear- array CCD detectors used with the better densitometers can distinguish adjacent features that are separated by 50 μm or greater (spatial resolution), which is more than adequate for most gel applications.

The better CCD cameras are cooled to increase their signal-to-noise ratios. They operate with illumination provided by either light boxes (UV or visible) for transmittance measurements or overhead lamps for epi-illumination. These devices are very versatile and they can acquire images from gels stained with colored or fluorescent dyes or silver. The epi-illumination feature allows CCD cameras to capture images of blots on opaque membranes (see the Application Focus on Blotting on this website). The spatial resolution obtainable with the cameras is entirely dependent on the properties of the lenses used and the area being imaged, but is generally in the 100-200 μm range. Their dynamic ranges for quantification often exceed four orders of magnitude.

Laser devices are the most sophisticated image acquisition tools. They are particularly useful for gels labeled with fluorescent dyes, since the lasers can be matched to the excitation wavelengths of the fluorophores. Detection is generally with photo- multiplier tubes. Some instruments incorporate storage phosphor screens for detection of radiolabeled and chemiluminescent compounds. Resolution depends on the scanning speed of the illumination module and can be as low as 50 μm.

An imager is the most significant investment of all electrophoresis apparatus. As with all significant purchases, comparison shopping among the available products is highly recommended. In practice, researchers access the data in their gels through the analysis software and it is the software that should be the primary consideration in any imaging system. Good software will be able to use data from most imaging devices. However, dedicated software designed for use with particular instruments provides the desirable feature of controlling the imagers with the software.

Software for 1-D gel analysis (Figure 1A) defines lanes and bands, quantifies bands, constructs standard curves and determines molecular weights. Images can be adjusted for contrast, processed in various ways, annotated, and exported to other files for publication or document control. 2-D analysis software (Figure 1B) defines and quantifies spots in 2-D PAGE gels. Those programs that use Gaussian spot modeling are better able to quantify proteins in overlapping spots than are the programs that define spots by contours. Programs for 2-D analysis include statistical software designed for quantitative comparisons of large numbers of gels. The programs are also set up for analysis of spot patterns derived from differentially expressed proteins and some can query databases to assist in protein identifications. They also can be used for image adjustments, annotation, and export in a variety of file forms.