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Keys to Successful Densitometry

Eagle Sight software aids image acquisition and processing

Michelle Cayouette *Michael Steege * Jane Moores
Stratagene Cloning Systems

Stratagenes new Eagle Sight software version 3.0 allows researchers to determine integrated density and concentration information from stained nucleic acids or proteins in gels. These functions provide a tool for quickly evaluating experimental data moments after an image has been acquired. To achieve more precise integrated density and concentration determinations, researchers using Eagle Sight software should understand how pixel saturation, background levels and analysis area can affect the accuracy of densitometry results.

The Eagle Eye II still video system is a digital imaging system that has been designed to document and analyze data from a wide variety of laboratory experiments. The newest version of the systems software, Eagle Sight software version 3.0, contains many features that aid image acquisition and processing.1,2 This version of Eagle Sight software can be used for determining molecular weight, integrated density and concentration information from applications such as stained nucleic acid gels or protein gels. The softwares densitometry functions are designed to provide results quickly and easily, even for researchers who have little experience working with the basic principles of densitometry. However, when the effects of pixel saturation, background levels and analysis area on densitometry calculations are understood, the analysis of integrated density and concentration will become more precise.

Integrated Density Determination

Integrated density is defined as the sum of pixel values minus a background value for each pixel within a bounded area. For a given area, increased brightness or size of a band or spot correlates to higher integrated density measurements. Integrated density measurements are useful for comparing band or spot intensities on images where no samples of known concentration (standards) are included. The Integrated Density function of Eagle Sight software version 3.0 allows rapid comparison between bands or spots within a single image from the Eagle Eye system.

figure 1

Determining integrated density with Eagle Sight software begins with choosing the Integrated Density function from the toolbar or Analyze menu (figure 1 (30k), RED). Integrated density information can then be viewed either by clicking directly on the band or spot or by clicking within a user-defined region of interest. This information can then be written on the image overlay, saved with the image and printed to preserve a hard copy of the final data.

Pixel Saturation: When acquiring an image for integrated density determinations, the user must keep the Saturation Warning function of the View menu enabled to avoid saturating pixels within bands or spots of interest. Enabling the Saturation Warning function results in image pixels with values of 255 to be displayed in red. Because 255 is the largest possible pixel value, increasing the exposure time of an image that already contains saturated pixels will not increase these pixel values further. However, pixel values below 255 will continue to increase with longer exposures until they too become saturated. Integrated density calculations on bands or spots having saturated pixels will produce measurements that are lower than expected, relative to nonsaturated samples. Therefore, spots or bands containing saturated pixels should not be used for precise densitometry analysis.

Background: The Integrated Density function sums all pixel values, minus a background value for each pixel, within an area of defined dimensions. Small differences in pixel value, even a value of 1 or 2, summed over an entire area can have a large cumulative effect on integrated density calculations. For this reason, care should be taken to have background values roughly equivalent throughout the analysis area and to properly define the background. Accurate densitometric analysis of images whose background levels vary greatly within analysis areas is difficult. In general, before beginning the analysis, the user should define the background level from an area of the image closest to the bands or spots of interest. This will ensure that the appropriate background value is subtracted from every pixel within the analysis area. If the user does not define the background, the software will automatically select the most common pixel value (the modal value) within the entire image and use this value for all background measurements.

Defining the background value before analysis can be accomplished either manually or automatically using the Set Background function located in the Analyze menu. By choosing the automatic option, users can define an area that they consider to be representative of background levels near bands of interest. Typically, this area will be right above or below the bands to be analyzed. In order to visualize background levels more readily, the False Color option from the Process menu can be used. By replacing the gradual grayscale with sharp differences in color, the False Color option displays small differences in pixel value more readily and allows the background threshold levels to be defined such that the most accurate results are provided. Once a region is defined, the software automatically calculates the modal value for this area and designates it as background. For more control, users can manually enter a numerical value for background after either viewing the pixel values in image areas near the bands of interest or viewing statistical information from defined regions. Both the Pixel Value and Statistics display functions are located in the View menu.

The size of the region of interest (ROI) is also likely to affect integrated density determinations. In general, the size of the analysis area should be only slightly larger than the bands or spots used for analyses. Analysis areas that are much larger than bands of interest have the potential to magnify errors due to small variances in background levels.

In order to compare integrated density measurements for determining relative intensities between bands or spots on the same image, sample areas should be kept the same size. If the dimensions of the areas are different, accurate analysis will be difficult unless the background has the same pixel value throughout the image analysis area. This is generally not the case. Preserving a rectangular analysis area of equivalent size throughout the analysis can be accomplished using the Region of Interest (ROI) function from the toolbar. The dimensions of the area can be preserved while the region is moved from one area of the image to another by simply (1) reselecting the ROI key from the toolbar, (2) moving the cursor until it is positioned over the previous ROI (this will cause the cursor to change to a hand symbol) and (3) dragging the ROI to a new position while holding down the left mouse button.

Concentration Analysis

Concentration can be determined directly from integrated density when standards of known concentration are analyzed along with samples of unknown concentration. Assuming that pixel values are not saturated, integrated density is directly proportional to concentration. Determining the concentration of an unknown sample is achieved by comparing the integrated density of that sample to a standard curve generated from the integrated densities and concentrations of known standards. For accurate determination of concentration values, users should take precautions so that pixels within bands of interest do not become saturated during acquisition and that the background is defined properly. After the image is acquired, determining the concentration of an unknown spot or band using Eagle Sight software version 3.0 begins by depressing the Concentration function key from the toolbar or Analyze menu (see figure 1 (30k),, GREEN). The software then prompts the user to set the background level. Defining an appropriate background level for concentration analysis entails the same steps as defining the background for integrated density analysis.

figure 2

Once a background level is set, the user can define standards either manually or automatically from the Concentration Calibration menu (figure 2 (27k),). In cases where accuracy is of the utmost importance, it may be necessary to manually detect standard bands in order to keep analysis areas identical throughout the procedure. Automatically defining standard bands is faster but can result in different sizes of analysis areas. After the standards have been defined, the standard curve can be plotted and viewed to determine how well the curve fits the data. If the default curve does not fit the standard data points well, the user can change the equation type from the Concentration Calibration menu until the equation that best fits the data is displayed. Determining the best equation is important because the software will use this curve to calculate the concentration of unknown bands or spots. After the standard curve is calibrated, values for samples of unknown concentration can be determined by detecting a band area, again either automatically or manually using the ROI function. Integrated density and concentration information can then be added to the image overlay, stored with the image and printed (figure 1 (30k),, RED and GREEN).

Conclusions

Eagle Sight software version 3.0 can be used to determine integrated density and concentration information from images of stained gels or other laboratory experiments. By ensuring that pixels within bands or spots of interest are not saturated during acquisition and that background levels and analysis areas are appropriately defined prior to analysis, researchers will be able to increase the accuracy and consistency of densitometry results.

REFERENCES

  1. Steege, M., and Cayouette, M. (1996) Strategies 9: 53-56.
  2. Cayouette, M., and Hansen, C. (1996) Strategies 9: 56-57.

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