Brief Instructions for ImageJ

Brief instructions for Tissue Analysis of Plant Organs using ImageJ

Basic strategy for organ analysis:

I. Calibrate Image. Make Sure Global Radio Button is set.
II. Create Masks

II A. Use Polygon Tool to Outline Entire Organ or Individual Tissues/Areas that you want to measure.
II B. Edit>Selection>Fit Spline (to smooth things out)
II C. Edit>Selection>Create Mask ( generate an outline, image of this to document/define what Tissue/Area you measure) Make sure you save this as a jpg file with a unique name!

III. Theshold Masks

III A. Open a Mask Image
III B. Image>Adjust>Theshold (0-125 seems to work well on masks created above) to theshold the tissue/area to be measured

IV. Make Area Measurements

IV A. Analyze>Set Measurements (Select measurements you want to collect)
IV B. Analyze>Analyze Particles (Set Analyze Radio Buttons: "Display Results" and "Add to Manager" to collect all measurements in one excel results sheet and display outlines of all areas measured in one overlay)
IV C. Make sure you save your results excel sheet under a unique name for further processing!

V. Use basic arithmatic relationships between the areas you have measured to calculate the absolute and relative tissue areas you wish to present in your final lab exercises.

Calibration Image:
Analyze/Set Scale... Use this dialog to define the spatial scale of the active image so measurement results can be presented in calibrated units, such as millimeters.
1. Before using this command, use the straight line selection tool to make a line selection that corresponds to known distance.
2. Then, bring up the Set Scale dialog, enter the known distance and unit of measurement, then click OK. ImageJ will have automatically filled in the Distance in Pixels field based on the length of the line selection.

Create Masks
Use Polygon Tool to Outline Entire Organ or Individual Tissues, that you want to measure.

Edit>Selection>Fit Spline (to smooth things out)

Edit>Selection>Create Mask ( generate an outline, image of this to incorporate, document what Tissue you measured in your exercise)

Make sure you save this Mask with a unique name!

Theshold Masks

Open a Mask Image

Image>Adjust>Theshold (0-125 seems to work well on masks created above) to theshold the tissue/area to be measured

Make Area Measurements

Analyze>Set Measurements (Select measurements you want to collect)

Analyze>Analyze Particles (Set Analyze Radio Buttons: "Display Results" and "Add to Manager" to collect all measurements in one excel results sheet and display outlines of all areas measured in one overlay)

Note to collect an image like the one above, you have to use SnagIt to capture the displayed window.

Make sure you save your results excel sheet under a unique name for further processing!

Use basic arithmatic relationships between the areas you have measured to calculate the absolute and relative tissue areas you wish to present in your final lab exercises.

Linear Measurements:

Use the Analyze>Set Measurements command to specify what area statistics are recorded.

With line selections, the following parameters can be recorded: length, angle (straight lines only), mean, standard deviation, mode, min, max and bounding rectangle (v1.34l or later). The mean, standard deviation, etc. are calculated from the values of the pixels along the line.

Select Straight Line, Drag and drop a line to correspond with region to be measured. Then click Analyze / Measure. Results are displayed in Results Window.
Repeat for as many regions you want to measure.

Once you have collected all your measurements, click File / Save As/ and save the data as an excel sheet. NB. It might help if you edit the data in the spreadsheet to keep track of tissues, etc. that have been measured.

Semiauto or auto measurements of cells:

Use Image / Color / Split Channels to make 3 gray Scale Images from your RGB image
Pick the Gray scale image that best delimits the cells of interest
Theshold Gray Levels that correspond towhat you want to measure: Image / Adjust / Theshold

Selections can be saved to disk using File/Save As/ROI and restored using File/Open. Multiple selections can be saved by transferring them to a blank window, drawing each one using Edit/Draw, and saving the resulting image. To restore one of the selections, open the image and click to the left of one of the drawn outlines with the wand tool.

Measure

Based on the selection type, calculates and displays either area statistics, line lengths and angles, or point coordinates. Area statistics are calculated if there is no selection or if a subregion of the image has been selected using one of the first four tools in the tool bar. Calculates line length and angle if a line selection has been created using one of the three line selection tools. Records coordinates if one or more points have been defined using the point selection tool. Use the Analyze>Set Measurements command to specify what area statistics are recorded. [Measurements]

With RGB images, results are calculated using brightness values. RGB pixels are converted to brightness values using the formula V=(R+G+B)/3, or V=0.299R+0.587G+0.114B if "Weighted RGB Conversions" is checked in Edit>Option>Conversions. The default weighting factors are the ones used to convert to from RGB to YUV, the color encoding system used for analog television. The weighting factors can be changed using the setRGBWeights macro function.

With line selections, the following parameters can be recorded: length, angle (straight lines only), mean, standard deviation, mode, min, max and bounding rectangle (v1.34l or later). The mean, standard deviation, etc. are calculated from the values of the pixels along the line.

To export the measurements as a tab-delimited text file, select File>Save As>Measurements from the ImageJ menu bar or File>Save As from the "Results" window menu bar. Copy the measurements to the clipboard by selecting Edit>Copy All from the "Results" window menu bar. You can also save measurements by right-clicking in the Results window and selecting Save As or Copy All from the popup menu.

The width of the columns in the "Results" window can be adjusted by clicking on and dragging the vertical lines that separate the column headings.

Analyze Particles...

This command counts and measures objects in binary or thresholded images. It works by scanning the image or selection until it finds the edge of an object. It then outlines the object using the wand tool, measures it using the Measure command, fills it to make it invisible, then resumes scanning until it reaches the end of the image or selection. Press the esc key to abort this process. Use Image>Adjust>Threshold to threshold an image. [Example]

Use the dialog box to configure the particle analyzer. Particles outside the range specified in the Size field are ignored. Enter a single value in Size and particles smaller than that value are ignored. Particles with circularity values outside the range specified in the Circularity field are also ignored. The formula for circularity is 4pi(area/perimeter^2). A value of 1.0 indicates a perfect circle. Note that the Circularity field was added in ImageJ 1.35e.

Select Outlines from the "Show:" popup menu and ImageJ will open a window containing numbered outlines of the measured particles. Select Masks to display filled outlines of the measured particles or Ellipses to display the best fit ellipse of each measured particles.

Check Display results to have the measurements for each particle displayed in the "Results" window. Check Clear Results to erase any previous measurement results. Check Summarize to display, in a separate window, the particle count, total particle area, average particle size, and area fraction. Check Exclude on Edges to ignore particles touching the edge of the image or selection.

Check Flood Fill and ImageJ will define the extent of each particle by flood filling instead of by tracing the edge of the particle using the equivalent of the wand tool. Use this option to exclude interior holes and to measure particles enclosed by other particles. The following example image contains particles with holes and particles inside of other particles.

The Record Starts option allows plugins and macros to recreate particle outlines using the doWand(x,y) function. The CircularParticles macro demonstrates how to use this feature.

Summarize

For each column in the results table, calculates and displays the mean, standard deviation, minimum and maximum of the values in that column.

Clear Results

Erases the results table and resets the measurement counter.

Set Measurements...

Use this dialog box to specify which measurements are recorded by Analyze>Measure and Analyze>Analyze Particles. [Measurements]

Area - Area of selection in square pixels. Area is in calibrated units, such as square millimeters, if Analyze>Set Scale was used to spatially calibrate the image.
Mean Gray Value - 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 Analyze>Calibrate was used to calibrate the image. For RGB images, the mean is calulated by converting each pixel to grayscale using the formula gray=0.299red+0.587green+0.114blue or the formula gray=(red+green+blue)/3 if "Unweighted RGB to Grayscale Conversion" is checked in Edit>Options>Conversions.
Standard Deviation- Standard deviation of the gray values used to generate the mean gray value.
Modal Gray Value - Most frequently occurring gray value within the selection. Corresponds to the highest peak in the histogram.
Min & Max Gray Level - Minimum and maximum gray values within the selection.
Centroid - The center point of the selection. This is the average of the x and y coordinates of all of the pixels in the image or selection. Uses the X and Y Results table headings.
Center of Mass - This is the brightness-weighted average of the x and y coordinates all pixels in the image or selection. Uses the XM and YM headings. These coordinates are the first order spatial moments.
Perimeter - The length of the outside boundary of the selection.
Bounding Rectangle - The smallest rectangle enclosing the selection. Uses the headings BX, BY, Width and Height, where BX and BY are the coordinates of the upper left corner of the rectangle.
Fit Ellipse - Fit an ellipse to the selection. Uses the headings Major, Minor and Angle. Major and Minor are the primary and seconday axis of the best fitting ellipse. Angle is the angle between the primary axis and a line parallel to the x-axis of the image. Note that ImageJ cannot calculate the major and minor axis lengths if Pixel Aspect Ratio in the Set Scale dialog is not 1.0.
Circularity - 4pi(area/perimeter^2). A value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated polygon. Values may not be valid for very small particles.
Feret's Diameter - The longest distance between any two points along the selection boundary. Also known as the caliper length. The Feret's Diameter macro will draw the Feret's Diameter of the current selection on the image.
Integrated Density - The sum of the values of the pixels in the image or selection. This is equavalent to the product of Area and Mean Gray Value. The Dot Blot Analysis example demonstrates how to use this option to analyze a dot blot assay.
Median- The median value of the pixels in the image or selection.
Skewness- The third order moment about the mean. The documentation for the Moment Calculator plugin explains how to interpret spatial moments.
Kurtosis- The fourth order moment about the mean.
Area Fraction- The percentage of pixels in the image or selection that have been highlighted in red using Image>Adjust>Threshold. For non-thresholded images, the percentage of non-zero pixels.
Limit to Threshold - If checked, only thresholded pixels are included in measurement calculations. Use Image>Adjust>Threshold to set the threshold limits.
Display Label - If checked, the image name and slice number (for stacks) are recoded in the first column of the results table.
Invert Y Coordinates - If checked, the XY origin is assumed to be the lower left corner of the image window instead of the upper left corner.
Redirect To - The image selected from this popup menu will be used as the target for statistical calculations done by the Measure and Analyze Particles commands. The Redirect To feature allows you to outline a structure on one image and measure the intensity of the corresponding region in another image. With ImageJ 1.35d or later this feature also works with stacks.
Decimal Places - This is the number of digits to the right of the decimal point in real numbers displayed in the results table and in histogram windows.