Analyze Menu

Measure

Computes the area and mean gray value of the current selection (or length if it's a line selection) and displays the results in the Info and Results windows. Use the Options dialog box to enable other measurements, such as perimeter. Use Undo to delete the last measurement or Delete Measurement to delete an arbitrary measurement. Only highlighted pixels are included in the computation if either density slicing or thresholding are enabled. The measurement counter is incremented by one each time you use Measure. Use Reset to reset it to zero. The default maximum number of measurements is 200, but this can be increased in the Options dialog box.

Use Show Results to display the list of measurement results, Print (with either the Info or Results window active) to print the results, Copy to copy results to the Clipboard, and Export to export results to a tab-delimeted (spreadsheet compatible) text file. There is a 32K byte limit when displaying, printing, or copying results, but no limit to the number of measurements that can be exported to a text file.

Calculated areas are displayed in calibrated units, such as square millimeters, if the Set Scale command has been used to establish the spatial scale. Density readings are displayed in calibrated units, such as optical density or isotope concentration, if the Calibrate command has been used to perform density calibration.

The most efficient way to organize measurement results is to have a spreadsheet loaded at the same time you are using Image . Make a series of measurements (command-1), Copy them to the Clipboard (command-C), click on the spreadsheet to activate it, select the cell where you want the measurements stored, Paste the results (command-V), click on the image window to reactivate Image , and finally, Reset the measurement counter (command-3).

Analyze Particles

Analyze Particles automatically counts and measures objects in binary or thresholded images. It does this by scanning across the image until it finds the boundary of an object, outlines the object using the same outlining routine used by the wand tool, measures the object using the equivalent of the Measure command, and then redraws the object in a different gray level so that it becomes invisible to the scanning process. Use Show Results to display the recorded measurements. Up to 32767 particles can be counted, but measurement recording is limited to the first Max Measurements (set in the Options dialog box) particles. Hold down the option key to bypass the dialog box. Use command-period to abort particle analysis.

Particles smaller than Min Particles Size or larger than Max Particle Size will be ignored. Note that the sizes must be specified in pixels. Checking Label Particles causes Analyze Particles to draw a number, corresponding to the measurement number, on each particle found. Check Outline Particles if you want particles to be outlined (this currently only works for density sliced images). Checking Ignore Particles Touching Edge causes Analyze Particles to ignore particles touching the edge of the image. Interior holes in area and mean density computations are included if Include Interior Holes is checked.

Show Results

Opens a window and displays the current results table in it. If the Results window is already open, then it is activated. No more than 32K characters can be displayed in the Results window, but there is no limit to the number of measurements that can be exported to a text file.

Use the Export command to save the measurements as a tab delimited text file that can be opened by many Mac programs, including Excel, StatView and KaleidaGraph. Or, as a shortcut, type command-S when the Results window is the active. For correct display, it may be necessary to adjust tab settings when opening an exported file with a word processing program such as Microsoft Word. Hold down the option key (or check Headings in Options) when Exporting (or Copying) results to include column and row headers. Use Print (command-P) to print the measurements, and Copy (command-C) to copy them to the Clipboard.

Show Histogram

Displays a plot showing the distribution of gray values within the selection. The plot shows, for each of the possible 256 gray values, the number of pixels in the selection that have that gray value. When the cursor is over the Histogram window, X (the gray value) and Y (the number of pixel with that gray value) are displayed dynamically in the Info window. Note that the Histogram command works with non-rectangular selections.

The Copy command, when the histogram window is the active window, copies both the histogram plot (as a PICT) and the histogram data values (as a single column of text) to the clipboard. Histogram data values can also be saved to a text file using the Export command.

Plot Profile

Generates a density profile plot based on the current rectangular or line selection. For rectangular selections wider than they are tall (or if the option key is down), it produces a "column average plot", where the width of the plot is equal to the width of the selection and each point in the plot represents the average gray value of the pixels in the corresponding column in the selection. For rectangular selections higher than they are wide, it does a top to bottom "row average plot", where the width of the plot is equal to the height of the selection and each point in the plot is the average of the corresponding row.

For line selections, it displays a plot of the gray values along the selected line. Pixel averaging is performed when using straight line selections and the line width is greater than one. For example, assuming the maximum line width is selected by clicking on the thickest line at the bottom of the tool palette, each data point plotted is the average of eight pixels.

The Profile Plot Options command can be used to vary the way profile plots are displayed. Use the Copy command to copy both the profile plot (as a PICT) and the data values (as a single column of text) to the clipboard. Plot values can also be saved to a text file using the Export command.

Surface Plot

Generates a surface plot of the current selection. The entire image is plotted if there is no selection. Type command-period to abort plotting. You can exaggerate the vertical scale in the plot by increasing the contrast of the image before plotting using the controls in the Map window. To improve the quality of plots of small selections, use the Scale and Rotate command to enlarge the selection before plotting.

Options

The Options dialog box allows you to specify which measurements are recorded by the Measure and Analyze Particles commands and displayed by Show Results. Double-clicking on the polygon tool also brings up this dialog box.

Area - Area of selection in pixels. Area is in calibrated units, such as square millimeters, if Set Scale has been used to spatially calibrate the image.

Mean - Average gray value within the selection. This is the sum of the gray values of all the pixels in the selection divided by the number of pixels. Reported in calibrated units (e.g., optical density) if the Calibrate command has been used to calibrate the image.

Standard Deviation - Standard deviation of the gray values used to generate the mean gray value.

X-Y Center - Center of the best fitting ellipse, measured from either the upper left or lower right corner of the image, depending on the status of the Invert Y Coordinates check box in the Preferences dialog box. This is the geometric center of the selection, not the density weighted center. This option is automatically enabled when the cross hair tool is used.

Modal Value - Most frequently occurring gray value within the selection. Corresponds to the highest peak in the histogram.

Perimeter/Length - Length around the outside of the selection, or line length for line selections. The perimeter is not computed for composite selections created using the control and option keys. This option is automatically enabled when Measuring a line selection.

Major/Minor Axis - Lengths of the major and minor axes of the best fitting ellipse.

Angle - Angle between the major axis and a line parallel to the x-axis of the image, or an angle measured using the angle tool. This option is automatically enabled when the angle tool is used.

Integrated Density - Sum of the gray values in the selection, with background subtracted. Use to measure the size (volume) of spots or bands in electrophoresis gels. It is computed using the following formula:

IntegratedDensity = N * (Mean - Background)

where N is number of pixels in the selection, and Background is the modal gray value (most common pixel value) after smoothing the histogram. Note that this formula assumes that the background is lighter (has lower pixel values) than the object being measured. The background level may be computed incorrectly if there isn't a well defined peak in the histogram. This can happen if not enough background is included within the selection or the background is not very uniform.

Min/Max - Minimum and maximum gray values within the current selection.

User 1, User 2 - Add new columns to the results table that can be used by user written macros to record and display derived results. Several example macros in the file "Measurement Macros" display results using User 1 and User 2 .


When Redirect Sampling is checked, pixel data for calculating the mean gray value is taken from the corresponding area of a second image. Requires that exactly two image be open, and that they be the same size.

The wand tool includes interior holes in area and mean density computations if Include Interior Holes is checked. If Wand Auto-measure is checked, the Measure command is automatically invoked whenever an object is outlined using the wand tool. Adjust Areas is used by the Gel Plotting Macros to attempt to correct for the tendency of the wand tool to underestimate the size of small peaks by not counting pixels under the peak boundary. When this option is selected, the area is increased by 0.5*Perimeter + 1.5*AspectRatio, where AspectRatio is the width to height ratio of the selection's bounding rectangle.

If Headings is checked, column and row headings will be added to results Copied to the Clipboard or Exported to a text file.

Max Measurements specifies the size of the arrays used to hold results produced by the Measurement command, by Analyze Particles, and by the angle and cross hair tools. You must Quit, and restart Image before the change will take effect.

Field Width specifies the column width, in characters, for results displayed in the Results window, Printed, Copied to the Clipboard, or Exported. Digits to the Right of Decimal Point specifies the number of digits that follow the decimal point.


Set Scale

Performs spatial calibration so that results from length and area measurements are presented in calibrated units, such as millimeters. Before setting the scale, use the line selection tool to make a straight line selection that corresponds to known distance. Then, bring up the Set Scale dialog box, select a unit of measurement from the Units pop-up menu, and enter the known distance in Known Distance.

Setting Pixel Aspect Ratio to a value other than 1.0 enables support for different horizontal and vertical spatial scales, for example 100 pixels/cm horizontally and 95 pixels/cm vertically. Before setting the aspect ratio, calibrate to a known horizontal distance. Then enter the pixel aspect ratio into Pixel Aspect Ratio . To find the pixel aspect ratio, measure the width and height (in pixels) of a digitized object with a known 1:1 aspect ratio. The pixel aspect ratio is computed by dividing the width by the height. Note that only the horizontal scale is shown. Divide the horizontal scale by the aspect ratio to compute the vertical scale. Note that the Measure command sets the ellipse major and minor axis lengths to zero when the pixel aspect ratio is not one.

Once the scale is set, you can switch to other units of measurements, for example from inches to centimeters. If you know what the scale for an image is (e.g., 300 dpi), then that value can be directly entered into Scale . Select Pixels from the pop-up menu to disable spatial calibration. To use a unit of measurement that is not predefined, select Arbitrary Units and manually type in the unit. As a shortcut, double-click on the line selection tool to bring up the Set Scale dialog box. Note that Image displays a black diamond in the title bar of spatially calibrated images.

Calibrate

Use this dialog box to calibrates to a set of density standards, such radioactive isotope standards or a calibrated optical density step tablet (available from Kodak). Before calibrating, you must record the mean gray value of each of the digitized standards. First, use the Reset command to set the measurement counter to zero. Then use one of the selection tools and the Measure command to record the mean gray value of each of the standards. Note that the standards must be measured in order, starting with the lowest gray value (lightest) standard.

When you have finished making the measurements, select Calibrate to display the calibration dialog box. To calibrate the image, enter the actual values in the Known column, select a curve fitting method, enter the unit of measurement, and click OK. If the calibration curve is not satisfactory, bring up the Calibrate dialog box again and select a different curve fitting method.

Rodbard is a four parameter general curve fit function proposed by David Rodbard at NIH. The form of the equation is: y = (D) + (A - D) / (1 + (x/C)^B).

Selecting Uncalibrated OD causes Image to convert gray values to uncalibrated optical density values using the following function:

Uncalibrated OD = log10(255 / (255 - Gray Value))

You do not need to measure OD standards or enter known OD values to enable this feature.

Pressing Invert OD performs the following function on the entered (known) standards:

Inverted OD = -log10(1.000 - 10^(-Entered OD))

Optical densities are converted to transmission, subtracted from perfect transmission and converted back to OD yielding the reciprocal density. This function is of use to those who have a positive set of optical density standards but a photographic or other negative image. It is also of use to those who quantitate reflected light images but wish to specify results in terms of transmitted light. This is often the case in anatomical imaging. Entered optical densities must range from 0.00001 to 4.62.

The Save button allows you to save both measured and known values to a text file so they can be restored later using the Open button. If there are no measured values (count=0), Open restores both the measured and known values, otherwise, only the known values are restored.

If you hold down the option key when invoking Calibrate you will be allowed to change the measured values. This feature allows you to enter measurements acquired previously or acquired from another system.

Note that Image displays a white diamond in the title bar of density calibrated images.

Redo Measurement

Allows you to repeat a previous measurement. Simply enter the number for the measurement you want to repeat and it will be replaced the next time you use the Measure command. Redo only works for measurements made using the Measure command, not for measurements made with the angle or cross hair tools.

Delete Measurement

Deletes the specified measurement and renumbers any subsequent measurements.

Reset

Resets the measurement counter to zero.

Restore Selection

Returns the previous region or line selection to its original position. If a selection is currently active, this command restores the previous one. Can also be used to transfer a selection from one window to another.

Label Selection

Outlines (in white) the current selection and labels it with the measurement count. Can be used to keep track of which objects have been measured. Hold the option key down to draw (also in white) the best-fit ellipse. To draw the ellipse, you must have previously used the Measure command and have selected X-Y Center, Ellipse Major Axis or Ellipse Minor Axis in Options.

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