Contents

Measurement of Line Spacing

Clear Image and Noisy Image

If the lines have enough contrast, their spacing can be measured via the profile tool. This exercise uses two images of SRM484, a microscope line spacing standard. One is so noisy that much signal averaging is necessary, while the other one is usable as-is.

The Info window can be calibrated to real units using the Analyze -> Set Scale menu. Any length measurements (or area measurements) will be given in terms of micrometers as well as pixels. Quoting the NIH Image manual, p. 6: "Images can be calibrated to either spatial or density standards using the Set Scale and Calibrate commands respectively. To calibrate spatially, use the line selection tool to measure a known distance, then use Set Scale to specify a unit of measurement (e.g. millimeters) and enter the known distance."

 

Image

ImageJ

Note - I typed in "um".  ImageJ interpreted the units properly as micrometers.

Note that the distance in pixels corresponds to the length in the info when stretching the selection line. Now, measurements are calibrated in real spatial units, rather than just pixels.

Image 

 The x, y and values are from the last cursor pixel location on the image, and are not relevant to the measurement.

The Count - is the number of times the measure menu has been used - also not relevant here.

Pixels is the area of the image in pixels (256 x 256). The pixel is square and serves as both linear and area units.

Area is the area in pixels converted to micrometers.

Remaining parameters are not relevant here.

 

ImageJ

Area:  the area of the image in square micrometers.  Note the difference from the measurement to the left, because the calibration is slightly different.

   When dragging, the line is narrow, but afterwards, the actual thickness is shown by the marching ants lines. (Not applicable to ImageJ)

A plot window appears, showing the intensities along the line:

 

Image

ImageJ

X and Y values appear live as the cursor is moved on the plot.


This section not applicable to ImageJ.

Brigher areas have lower pixel values as the default. If you prefer to see the lines as peaks:

The plot will appear inverted (the old plot will disappear):


Noisy image

(I have enhanced the image contrast for visualizing here.)

This is the resulting profile. The positions of the lines are obscured by the noise - signal averaging with the fat line is not sufficient.

Image

ImageJ

 


This section not applicable to ImageJ.

where all of the lines can be seen. The peaks will be broadened if the line selection is not exactly perpendicular to the lines to be measured. The selection for the above plot looked like this:


 

Another approach is to rotate the image so that the lines are horizontal, then use the rectangle selection and plot the profile. The profile is taken along the longest side of the rectangle.

 

Here is another illusion - the lines may not look horizontal vertical, but they almost are. (Check by measuring their angle of inclination, or by noting the vertical allignment against the rectangle selection tool.)

Image

(Note the inches for the length units - that happened to be the default calibration unit when I took the profile. The units are therefore arbitrary.)

ImageJ

Note that the peaks are broader than the lines because the lines are not quite vertical.