Strip densitometry procedure

1. Make sure the image is grayscale before starting. Pixel values on color images may or may not have any correlation with brightness. Select ``Color...Colorgray scale'' to convert an image to grayscale.
2. Click ``Process..Strip densitometry''. The Strip densitometry dialog should appear.
3. Change the settings in the dialog box as desired. The most important is to specify whether the largest signal in your image is ``Black'' or ``White''. Click on ``Maximum signal=black'' or ``Maximum signal=white'' depending on whether the features of interest are darker or lighter than the background.
4. Note that there is no box for background subtraction in the dialog box. Background can be subtracted interactively or automatically in the graph. The graph is created automatically if ``Plot result'' is checked, and is automatically updated when you measure a new area.
5. Set the Width as desired if you are using ``Fixed width'' scanning. This value must be in pixels, because the algorithm is concentrating only on raw pixels at this point.
6. Click `Accept' to begin densitometry.
7. Use the mouse to select points on the image that define the boundaries of the region to measure. For Fixed with scanning, select one point at the beginning and one at the end of the region so that 2 boxes appear on the screen.. This region need not be vertical or horizontal, but can be at any angle.
8. For Rhomboid-type scanning, select two points at the start and two points at the end of the strip to scan. This will define two lines. Click once on each corner of the rectangular area to be scanned, in a clockwise direction, so that 4 boxes appear on the screen.
9. Drag the 2 or 4 control points using the mouse to the desired positions. If any of the boxes are in the wrong position, or the lines connecting them are crossed, click and drag the boxes to the correct position. (NOTE: if the lines are crossed, the algorithm automatically swaps the coordinates to un-cross them).
10. Press the space bar to begin densitometric measurement.
11. The image will be scanned between the two lines as shown in the figure below. A graph should appear showing the results. If the graph is off scale, click ``Rescale''.
    
    Direction of pixel scanning in various types of strip densitometry.
12. In the graph, you can subtract background (baseline) values manually or let the computer select a best fit line automatically. Once the feature of interest has been isolated as a single peak, drag on the graph with the mouse to highlight the peak. The portion of the graph that is selected will be calculated and the measured area will be shown in a small list box. This ``area'' represents the total signal minus background of the feature in the image, multiplied by the Calibration factor.
13. In the graph window, click on ``Save to disk'' to save the densitometry tracing in an ASCII file.
14. Select additional control points and press the space bar again to make additional measurements. The graph will automatically be updated and resized if necessary to accommodate the new region. It is not necessary to click `Accept' before taking a new measurement. You can continue selecting additional areas indefinitely.
15. When you are finished performing densitometric measurements, click the main Cancel button or press Esc. This will unmap the densitometry dialog box. You will be prompted to save the graph and the list box containing the results.

Options:

Pixel density calibration OD table If checked, this will cause imal to use the value in the optical density table in its calculations instead of the raw pixel value. This information is sometimes provided by digital scanners in the form of a `gray response curve' or `gamma curve' that is embedded in the TIFF file. For most images, this option will have no effect since no gray response curve is present. However, you can easily create a curve by clicking ``Config..Show OD table'' and modifying the graph to create a curve of any desired shape. Since this curve has only 256 elements, it is only applicable for 8-bit grayscale images. If you create a curve manually, it must be monotonically increasing or decreasing (i.e., no peaks).

z units If checked, this causes imal to use the calibrated pixel value in its calculations instead of the raw pixel value. Pixel value calibration is performed before starting densitometry, by clicking ``Process..Calibration'', checking the desired equation to which the `z value' is to be fitted, and then clicking on each calibration standard in the image (See Sec. 8.9) and entering the known value in the spreadsheet. imal performs a linear regression on the data and uses these coefficients to calibrate pixels in the image.

NOTE: For this option, the ``Maximum Signal Black/White'' setting is ignored, because the user calibration automatically defines what pixel value is the maximum signal.

None In this option, the signal and density are given as raw densities (expressed as a number between 0 and 1). (signal = density $\times$ number of pixels.)

Automatically save scan

If this option is checked, each scan will automatically be saved in an ASCII file.

Filename for scan

A default filename of ``1.scn'' is provided. If the 1st 8 characters of the filename consist entirely of digits, the program will automatically increment the filename after each scan (For example, if the starting filename was ``1000.dat'', subsequent scans would be saved under 1001.dat, 1002.dat, etc.). If the filename contains letters, the filename must be typed in each time.

Plot results

If checked, the scan results will automatically be plotted on the screen after each scan. Clicking the ``OK'' button on the graph makes the graph disappear upon the return to scanning mode. The graph is always automatically scaled in the Y direction to fill the entire box. When the graph is visible, the data can be saved to disk, a background curve can be subtracted from the data, or the graph can be captured into a new image (See ``Plotting densitometry results and other data'').

If the image has been calibrated, moving the cursor over the plot area of the graph causes two different x-values are printed. The upper x value is the data point number, and the lower x value is the calibrated value for the center pixel of the line along which the strip densitometry was performed. If the image has not been calibrated, the two x values will be identical. Clicking-and- dragging within the plot selects a portion of the graph for area calculation. The selected area is highlighted in reverse color. If the image has been calibrated, this area (printed at the lower right of the graph) is also calculated from the calibrated values.

Pause to show region

If checked, the program pauses after drawing the 4 boxes, allowing you to verify that you selected the correct region. Press a key to continue.

Fixed width Width to be used when ``Scan Type'' is set to ``Fixed width''.

Panel A in the figure below is a typical 8-bit image showing part of a nitrocellulose blot stained for proteins with colloidal gold. The dark area is the protein calexcitin. It is necessary to know the total amount of the protein in this band.

• The band was first measured using ``fixed-width scan'' in ``Strip densitometry''. The rectangle was oriented so that the band was perpendicular to the start and end points of the rectangle. This produced the trace shown in B.
• A manual baseline was subtracted, using the points shown in C (control points are shown by small squares). The program generated a Bezier curve and subtracted it from the data, producing D.
• The peak area can now be easily determined, by dragging the mouse to highlight the peak (E).

Densitometric analysis of a protein band using strip densitometry.

Despite the complicated statistical analysis available in spot densitometry (above), this method of visually identifying the band is still the most accurate and reproducible densitometric technique.

NOTE: Densitometry may not work correctly on zoomed images. Use ``Change size'' first.